Model DNA for investigation of mechanism of nucleotide excision repair
The living cell DNA is under permanent attack of a variety of exogenous and endogenous damaging factors. Nucleotide excision repair (NER) is main pathway which removes a wide variety of bulky DNA adducts formed by UV light, electrophilic environmental mutagens, and chemotherapeutic agents. NER proce...
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nasplib_isofts_kiev_ua-123456789-1543012025-02-23T17:51:16Z Model DNA for investigation of mechanism of nucleotide excision repair Модельні ДНК для дослідження механизму нуклеотидної ексцизійної репарації Модельные ДНК для исследования механизма нуклеотидной эксцизионной репарации Evdokimov, A.N. Lavrik, O.I. Petruseva, I.O. Reviews The living cell DNA is under permanent attack of a variety of exogenous and endogenous damaging factors. Nucleotide excision repair (NER) is main pathway which removes a wide variety of bulky DNA adducts formed by UV light, electrophilic environmental mutagens, and chemotherapeutic agents. NER process in mammalian cells consistently leads to the very specific excision of damaged DNA fragments 24–32 nucleotides in length. The following DNA repair synthesis and DNA ligation restore intact DNA helix. The main set of the genes inactivated in NER-deficient higher eukaryotic cells was identified; about 30 proteins are involved in the specific multi-subunit complexes responsible for NER process. The specific NER feature is wide substrate specificity and great difference of damages elimination efficiencies. A key limiting step in NER is damage recognition and verification. One of the advanced and upcoming approaches to NER process investigation is based on the application of model DNAs – artificial DNA structures, which are analogs of substrate or intermediates of the repair process. This article reviews our current knowledge concerning the model DNA design, synthesis and application as a tool for the NER process comprehensive study. ДНК живих клітин перебуває під постійним впливом різноманітних пошкоджуючих факторів екзо- і ендогенного походження. Нуклеотидна ексцизійна репарація (NER) видаляє з ДНК широкий набір об’ємних адуктів, які утворилися в результаті дії УФ опромінення, а також електрофільних речовин – забруднювачів довкілля, що чинять мутагенний вплив, та хіміопрепаратів. У процесі репарації, яку виконує система NER ссавців, відбувається специфічне вищеплювання з ДНК фрагментів розміром 24––32 нуклеотиди, що містять пошкодження. Подальший репаративний синтез і лігування ДНК відновлюють інтактність спіралі ДНК. Ідентифіковано гени, інактивовані в NER-дефіцитних клітинах вищих евкаріотів. В репарації беруть участь приблизно 30 білків, які формують специфічні мультисубодиничні комплекси. Система NER характеризується широкою субстратною специфичністю і при цьому великими розбіжностями в ефективності видалення пошкоджень. Ключовою лімітуючою стадією процесу є упізнавання та верифікація пошкоджен. До ефективних і таких, що розвиваються, підходів до вивчення процесу NER належить метод, заснований на використанні модельних ДНК – синтетичних структур, які є аналогами субстрата або інтермедіатів цього процесу. Розглянуто існуючі дані щодо способів конструювання модельних ДНК та застосування їх як інструмента для всебічного дослідження процесу NER. ДНК живых клеток находится под постоянным воздействием различных повреждающих факторов экзо- и эндогенного происхождения. Нуклеотидная эксцизионная репарация (NER) удаляет из ДНК широкий набор объемных аддуктов, образовавшихся в результате воздействия УФ облучения, а также электрофильных веществ – загрязнителей окружающей среды, оказывающих мутагенное действие, и химиопрепаратов. В процессе репарации, проводимой системой NER млекопитающих, происходит специфическое выщепление из ДНК фрагментов размером 24–32 нуклеотида, содержащих повреждения. Последующий репаративный синтез и лигирование ДНК восстанавливают интактность спирали ДНК. Идентифицированы гены, инактивированные в NER- дефицитных клетках высших эукариотов. В репарации участвуют примерно 30 белков, формирующих специфические многосубъединичные комплексы. Система NER характеризуется широкой субстратной специфичностью и при этом большими различиями в эффективности удаления повреждений. Ключевой лимитирующей стадией процесса является узнавание и верификация повреждения. К эффективным и развивающимся подходам к исследованию процесса NER принадлежит метод, основанный на использовании модельных ДНК –синтетических структур, являющихся аналогами субстрата или интермедиатов этого процесса. Рассмотрены существующие данные о способах конструирования модельных ДНК и применении их в качестве инструмента для всестороннего изучения процесса NER. 2014 Article Model DNA for investigation of mechanism of nucleotide excision repair / A.N. Evdokimov, O.I. Lavrik, I.O. Petruseva // Вiopolymers and Cell. — 2014. — Т. 30, № 3. — С. 167-183. — Бібліогр.: 107 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000893 https://nasplib.isofts.kiev.ua/handle/123456789/154301 577.21 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України |
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Reviews Reviews Evdokimov, A.N. Lavrik, O.I. Petruseva, I.O. Model DNA for investigation of mechanism of nucleotide excision repair Вiopolymers and Cell |
| description |
The living cell DNA is under permanent attack of a variety of exogenous and endogenous damaging factors. Nucleotide excision repair (NER) is main pathway which removes a wide variety of bulky DNA adducts formed by UV light, electrophilic environmental mutagens, and chemotherapeutic agents. NER process in mammalian cells consistently leads to the very specific excision of damaged DNA fragments 24–32 nucleotides in length. The following DNA repair synthesis and DNA ligation restore intact DNA helix. The main set of the genes inactivated in NER-deficient higher eukaryotic cells was identified; about 30 proteins are involved in the specific multi-subunit complexes responsible for NER process. The specific NER feature is wide substrate specificity and great difference of damages elimination efficiencies. A key limiting step in NER is damage recognition and verification. One of the advanced and upcoming approaches to NER process investigation is based on the application of model DNAs – artificial DNA structures, which are analogs of substrate or intermediates of the repair process. This article reviews our current knowledge concerning the model DNA design, synthesis and application as a tool for the NER process comprehensive study. |
| format |
Article |
| author |
Evdokimov, A.N. Lavrik, O.I. Petruseva, I.O. |
| author_facet |
Evdokimov, A.N. Lavrik, O.I. Petruseva, I.O. |
| author_sort |
Evdokimov, A.N. |
| title |
Model DNA for investigation of mechanism of nucleotide excision repair |
| title_short |
Model DNA for investigation of mechanism of nucleotide excision repair |
| title_full |
Model DNA for investigation of mechanism of nucleotide excision repair |
| title_fullStr |
Model DNA for investigation of mechanism of nucleotide excision repair |
| title_full_unstemmed |
Model DNA for investigation of mechanism of nucleotide excision repair |
| title_sort |
model dna for investigation of mechanism of nucleotide excision repair |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2014 |
| topic_facet |
Reviews |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/154301 |
| citation_txt |
Model DNA for investigation of mechanism of nucleotide excision repair / A.N. Evdokimov, O.I. Lavrik, I.O. Petruseva // Вiopolymers and Cell. — 2014. — Т. 30, № 3. — С. 167-183. — Бібліогр.: 107 назв. — англ. |
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Вiopolymers and Cell |
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2025-11-24T04:42:36Z |
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2025-11-24T04:42:36Z |
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| fulltext |
REVIEWS
UDC 577.21
Model DNA for investigation of mechanism
of nucleotide excision repair
A. N. Evdokimov1, 2, O. I. Lavrik1, 2, 3, I. O. Petruseva1
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
8, Akad. Lavrent’eva Ave., Novosibirsk, Russian Federation, 630090
2Altai State University, Ministry of Education and Science of the Russian Federation
61, Lenina Ave., Barnaul, Russian Federation, 656049
3Novosibirsk State University, Ministry of Education and Science of the Russian Federation
2, Pirogova Str., Novosibirsk, Russian Federation, 630090
irapetru@niboch.nsc.ru
The living cell DNA is under permanent attack of a variety of exogenous and endogenous damaging factors. Nuc-
leotide excision repair (NER) is main pathway which removes a wide variety of bulky DNA adducts formed by
UV light, electrophilic environmental mutagens, and chemotherapeutic agents. NER process in mammalian cells
consistently leads to the very specific excision of damaged DNA fragments 24–32 nucleotides in length. The fol-
lowing DNA repair synthesis and DNA ligation restore intact DNA helix. The main set of the genes inactivated in
NER-deficient higher eukaryotic cells was identified; about 30 proteins are involved in the specific multi-subunit
complexes responsible for NER process. The specific NER feature is wide substrate specificity and great diffe-
rence of damages elimination efficiencies. A key limiting step in NER is damage recognition and verification.
One of the advanced and upcoming approaches to NER process investigation is based on the application of mo-
del DNAs – artificial DNA structures, which are analogs of substrate or intermediates of the repair process. This
article reviews our current knowledge concerning the model DNA design, synthesis and application as a tool for
the NER process comprehensive study.
Keywords: nucleotide excision repair, bulky DNA adducts, model DNA.
Introduction. The DNA of the living cells is subject of
various modifications due to the impact of both exoge-
nous and endogenous factors. There are several ways
of damaged DNA repair in order to prevent the accu-
mulation of modifications and to protect the genetic
information in cells. The nucleotide excision repair
(NER) is one of the most important ways to achieve ge-
nome safety. NER protects the cells from the broadest
range of structurally and chemically different bulky
DNA damages. The damages are present in the form of
modifica tions of nitrogenous bases, occurring due to
the effect of UV light, ionizing radiation, environmen-
tal electrophilic chemical mutagens, some medical drugs
as well as chemically active endogenous metabolites,
including reactive derivatives of oxygen and nitrogen.
In the cells of higher eukaryotes the NER system with
high precision removes oligonucleotide fragments 24–
32 nucleotides in length, containing the damage [1].
The subsequent reparative synthesis using the non-da-
maged strand as a template, followed by the ligation of
the single-strand break, complete the process of DNA
repair [2] (Fig. 1). The main genes, inactivated in NER-
deficient cells of higher eukaryotes, have been identi-
fied and the protein factors and enzymes have been de-
termined. This process is known to be performed by co-
ordinated activity of about 30 proteins, subsequently
forming on DNA complexes of variable composition
[3]. The mutations in the genes, encoding these pro-
teins, lead to the down regulation of the NER process.
Such mutations result in some diseases, including xe-
167
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 3. P. 167–183 doi: http://dx.doi.org/10.7124/bc.000893
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
roderma pigmentosum (XP), Cockayne syndrome (CS)
and trichothiodystrophy (TTD), characterized by UV-
sensitivity, high risk of cancer and a number of neuro-
degenerative symptoms [4].
There are two variants of NER process, differing at
the damage recognition step. The signal for the start of
the transcription-coupled repair (TCR) is the stop of
RNA-polymerase, when the damage in the transcribed
DNA strand arrests the transcription activity [5, 6]. Glo-
bal genome nucleotide excision repair (GG-NER) per-
forms the searching and removal of the bulky damages
regardless of the functional state of the genome, inclu-
ding the non-transcribed parts of chromatin. The specia-
lized sensor factors – XPC (protein complex XPC/
HR23B/Cen2) and XPE (DDB1/DDB2 heterodimer,
promoting the recognition of UV-damage) are respon-
sible for the primary recognition of the damage in the
GG-NER process [1, 7–10].
A broad range of biochemical and molecular bio-
logy methods, including the methods, based on the ap-
plication of artificial model DNAs, are used for the de-
tailed study of the mechanism of the interaction bet-
ween NER protein factors among themselves and with
the DNA in vitro.
Generally the model DNAs are either the analogues
of the substrate (of the damaged DNA) or the analogues
168
EVDOKIMOV A. N. ET AL.
DNA damage
XPC-HR23B
TFIIH
RPA
XPG
XPC-HR23B
XPA
ERCC1-XPF
ERCC1-XPF
RFC
PCNA
Pol ä
Pol ä
RFC
PCNA
TFIIH XPG
XPA
RPA
Ligase I
or
Ligase III
Undamaged DNA
Ligase I
or
Ligase III
Fig. 1. Scheme of global genome nucleotide excision repair
of the intermediates of the NER process. In particular,
these intermediates are modified duplexes, containing
artificially introduced non-complementary parts («bub-
bles» of different length), «fork»-like structures, as well
as some structures, containing 5' or 3'-overhangs [11–14].
The main types of damages, repaired by the NER
system and the modifications, used for model DNA
synthesis. Regardless of the fact that the model DNA
has been used to study the NER process for many years,
the elaboration and improvement of the methods for the
model DNA synthesis still remains actual. Different
bulky lesions obtained due to the modification of nitro-
genous bases are used as a damage-mimicking adducts,
repaired by the NER system. Such adducts may imitate
the structure of damages that appear in cellular DNA un-
der exogenous or endogenous damaging factors. On
the other hand, these lesions may be artificial construct-
ions, the introduction of which into dsDNA also leads to
the distortion in its structure and the change in the ther-
modynamic stability.
The products of UV irradiation. One of the main
DNA-damaging agents is UV radiation. It was determi-
ned that UV light is the most dangerous for the organism
part of the sunlight, reaching the surface of the Earth.
The UV spectrum is composed of three parts: UV A
(300–400 nm), UV B (290–320 nm) and UV C (200–
290 nm) with the highest energy, but it is mainly filte-
red out by the ozone layer of the Earth. The maximal
formation of the photoproducts is observed when DNA
is exposed to UV light at the wavelength lesser than
300 nm. It is in good agreement with the adsorption
spectrum of the main DNA chromophores – thymine
and cytosine. Prokaryotes and lower eukaryotes are ca-
pable of remove UV-damages using the NER system.
Additionally, they can repair UV-lesions by the specific
enzymes – photolyases, transforming pyrimidine dimers
into the intact monomers [15]. In contrast, in higher
eukaryotes cells UV-damages only can be removed by
NER system.
Pyrimidine dimers (Fig. 2), the UV-radiation pro-
ducts, are the most common model of pyrimidine da-
mage.
Cyclobutane pyrimidine dimers (CPD) (Fig. 2, b)
are formed due to (2 + 2) cyclo attaching along double
bonds C5–C6 of adjacent pyrimidine bases. The majo-
rity of CPD are formed by adjacent TT bases, but the
formation of these adducts is also possible due to the in-
teraction of CC, CT or TC depending on the wavelength,
radiation dose and nucleotide sequence. The formation
of CPD is reversible after the impact of UV-light at the
wavelength of 250 nm [16].
6–4-photoproducts (Fig. 2, a) are included in the
number of the most common UV-damages in the cell
[17]. The intermediate, obtained due to (2 + 2) attaching,
is regrouped with the formation of 6–4-photoproduct
which with further radiation may be reversed into the
Dewar isomer. In contrast with CPD, 6–4-photopro-
ducts cause higher distortion of the DNA structure with
the disturbance of nucleotide coupling at the site of the
damage, which is reflected in higher efficiency of their
repair by the NER system [18, 19].
The products of environmental chemical agents. Po-
lycyclic aromatic hydrocarbons (PAH) are widespread
environmental pollutants, which are the products of in-
complete combustion of different materials. Benzo[a]-
pyrene and benzo[c]-anthracene are inert non-polar
compounds. However in the process of metabolic
detoxification in the mammals, involving cytochrome
C and epoxyhydrolases action, potentially toxic lipophilic
PAH molecules transform not only into excreted soluble
derivatives, phenols and dihydrodioles, but also into
electrophilic, chemically active diolepoxides, capable to
stereoselective reacting with nucleobases with the for-
mation of bulky adducts [20]. The repair of benzo[a]-
pyrene damages has been studied in details. The me-
thods of synthesis of the DNA, containing dG- and dA-
benzo[a]-pyrene adducts with different structure and
stereochemistry, are developed. Analysis of repair pro-
cess of such model DNA, along with the results of in-
strumental methods of analysis and computer simula-
169
MODEL DNA FOR INVESTIGATION OF MECHANISM OF NUCLEOTIDE EXCISION REPAIR
UV
a b
O
HN
N
O
O
NH
N
O
O
HN
N
O
O
H
N
N
O
HN
N
O
O
NH
N
O
OH
Fig. 2. DNA UV irradiation products – pyrimidines dimers: a –
6–4-photoproducts; b – cyclobutane pyrimidines dimers
tion led to an understanding of the principles of the
bulky damages recognizing by the NER system. The im-
pact of the stereochemistry, thermodynamic properties
of such DNAs, sequence of damaged DNAs as well as
the adduct topology, on the efficiency of lesion recog-
nition and removal from DNA have been considered in
a fine details [20–24].
The metabolic transformation of the polycyclic di-
hydrofuranes of aflatoxin B1 (AFB1), aflatoxin G1 and
sterigmatomycin, which are produced by some kinds of
fungi, polluting food products, also involves the forma-
tion of the corresponding epoxides [25], which interact
with DNA.
Another class of compounds, damaging mammali-
an DNA, is arylamines (Fig. 3). Arylamines are also pre-
sented in the tobacco smoke, hair dye and some other
sources. Tobacco smoke contains some compounds of
the arylamine class, which are known carcinogens, in
particular, 2-naphthylamine, 4-aminobiphenyl and ben-
zidine [26] (Fig. 3, a) as well as aminofluorene and ace-
tylaminofluorene. Arylamines may be activated in the
process of metabolism in N-hydroxy-, N-acetoxy- or
N-sulfoxy compound. Further heterolytic breakage of
N-O bond leads to the formation of highly active inter-
mediates, forming the extensive adducts during the in-
teraction with DNA [27] (Fig. 3, b). Many works,
dedicated to the investigation of the NER mechanism
and mutagenesis processes, describe the application of
the model DNA, containing N-(deoxyguanosin-8-yl)-
2-acetylaminofluorene (dGAAF) [28–30]. Another
threat to the mammalians is presented by the nitro
compounds of the polycyclic aromatic hydrocarbons.
These compounds released into the environment from
different sources; mainly these are the products of in-
complete combustion, but some are also present in food
and drinks. 6-nitrochrysene (6-NC) is not the most
common compound of this class, but it is also known as
a carcinogen, capable to induce breast cancer. The car-
cinogenic effect of 6-NC exceeds not only that of ben-
zo[a]-pyrene, but also such a powerful carcinogen as he-
terocyclic aromatic amine 2-amino-1-methyl-6-phenyl-
imidazol[4,5-b]pyrimidine [31]. It is commonly known
that the metabolic activation of 6-nitrochrysene may
occur in one of two ways (Fig. 4). The first way sug-
gests the ordinary reduction of the nitrogroup with the
formation of 6-hydroxylaminochrysene, the interaction
of which with deoxyguanosin in DNA leads to the for-
mation of N-(deoxyguanosin-8-yl)-6-aminochrysene
and 5-(deoxyguanosin-N(2)-yl)-6-aminochrysene [32,
33]. The second way is the oxidation of the fourth ring of
chrysene, the reduction of a nitrogroup with the forma-
tion of high-reactive electrophilic intermediate trans-1,
2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene.The
interaction of this intermediate with DNA also leads to
170
EVDOKIMOV A. N. ET AL.
N
H
N
NH
N
N
O
HN
O
NH2N
NH
N
N
O
N
O
N
O
O
O
NH2N
NH
N
N
O
NH
DNA
NH2
NH2 NH2H2N
a
b
Fig. 3. a – the most
common arylamines
(left to right): naph-
thylamine, 4-aminobi-
phenyl and benzidine;
b – interaction of N-
acetoxyacetylamino-
fluorene with DNA le-
ads to bulky adducts
formation: 3-(deoxy-
guanosine-N2-yl)-2-
acetylaminofluorene
(left), N-(deoxyguano-
sine-8-yl)-2-acetyl-
aminofluorene (mid-
dle) or N-(deoxygua-
nosine-8-yl)-2-amino-
fluorene (right)
the formation of 5-(deoxyguanosin-N(2)-yl)-6-amino-
chrysene [34, 35]. It has been recently demon- strated
that the repair of these damages occurs with low effici-
ency (approximately the same with repair of N2-deoxy-
guanosin-benzo[a]-pyrenyl adducts) [36], i. e. one order
less efficiently than the repair of efficient NER subst-
rates – intrastrand crosslinks, introduced into dsDNA
due to the effect of platinum derivatives [37, 38].
The effects of the medical preparations on DNA
components. Cis-diaminodichloroplatinum (II) (or cis-
platin) is one of commonly used chemotherapeutic drug,
performing its cytotoxic function via the DNA dama-
ging. Cisplatin is a non-charged complex of bivalent
platinum with the configuration of a flat square. Once
intravenously injected into the organism, this complex
compound remains stable in the blood plasma until it
penetrates the cell cytoplasm, where low concentration
of chloride ions leads to their replacement in the inner
sphere of the complex with water or hydroxy-ions with
the formation of the active electrophilic agent. Its in-
teraction with DNA leads to the formation of a monoad-
duct as well as intrastrand or interstrand crosslinks. Si-
milar to the UV-lesions, the efficiency of repair of the
cisplatin-induced damages is affected by the differen-
ces in their structure [39,40]. An example of the inter-
action between the pharmacological activity of the sub-
stance and its structure is bifunctional platinum anti-
cancer drugs. As stated above, pharmacologically acti-
ve substances are platinum complexes with the cis-struc-
ture [39, 40], whereas the trans-isomer (transplatin) has
no clinical efficiency. However the complexes with the
trans-structure, demonstrating higher cytotoxicity in the
tumor cells, have been obtained. The replacement of an
amino group in transplatin for an iminoester resulted in
a considerable increase in the complex cytotoxicity even
in comparison with the cytotoxicity of the correspon-
ding cis-isomer. The trans-(PtCl2(E-iminoester)2 de-
monstrates high anticancer activity regarding cancer
cells, resistant to the effect of commonly used platinum
compounds [41]. There have been attempts of elabora-
ting a new generation of antineoplastic preparations,
based on platinum II, in order to solve the problem of
drug resistance and decreasing the side effects of che-
motherapy [42]. The monofunctional complexes of pla-
tinum II of the general formula cis-[Pt(NH3)2(N-hetero-
cycle)Cl]Cl form a single covalent bond with DNA, in-
troduce minor distortions into the regular structure of
dsDNA and differ in their properties from classic rea-
gents like cisplatin. Pyriplatin (a monofunctional comp-
lex with pyridine as a ligand) may serve as an example
of such drug. The variation of heterocyclic replace-
ments allowed revealing the compounds with high an-
titumor activity [43]. Phenanthriplatin, containing an
extensive ligand phenanthridin (cis-[Pt(NH3)2(phenan-
tridin) Cl]NO3), has a considerably higher activity than
commonly used cisplatin and oxaliplatin. There are al-
so differences in the spectrum of antitumor activity, de-
monstrated by this compound, which means that phe-
nanthriplatin may be active regarding the types of can-
cer cells, demonstrating resistance to common platinum
chemotherapy [44].
In recent years the attention of researchers was at-
tracted to bulky adducts, introduced in DNA due to inter-
action of exocyclic amino groups of DNA purine bases
with reactive metabolites of aristolochic acids (MAA).
171
MODEL DNA FOR INVESTIGATION OF MECHANISM OF NUCLEOTIDE EXCISION REPAIR
NO2
NHOH
NHOH
HO
OH
NHN
NH
N
N
O
NHOH
OH
HO
NH2N
NH
N
N
O
NH
DNADNA
trans dihydroxy
ihydro hydroxylamine chrysene
-1,2- -1,2-
d -6-
5-( -N(2)- )
-
deoxyguanosine yl
6-aminochrysene
N-( -8- )-
6-
deoxyguanosine yl
aminochrysene
6-hydroxylamine chrysene
Fig. 4. 6-nitrochrysene activation leads to bulky adducts
It has been recently revealed that these components of
herbal medical drugs, used for centuries, have serious
toxic effect and cause nephropathy, in 50 % leading to
cancer [45].
The results of the research, performed using the mo-
del DNA, demonstrated that the newly formed MAA
derivatives of deoxyguanosine are removed from DNA,
whereas the adducts of deoxyadenosine are accumula-
ted in the organism which leads to the progress of di-
seases [46].
Also has been described the application of the mo-
del structures, containing modifications, which introdu-
ced into DNA due to the effect of hormonal drugs like
premarin. The metabolites of the conjugated estrogens,
4-hydroxyequilenin and 4-hydroxyequilin, are widely
used as a components of drugs for the hormone-repla-
cement therapy. They are capable of autoxidation with
the formation of o-quinones, actively interacting with
the nucleobases of DNA, mainly with cytosine, which
results in the formation of bulky adducts that are hard to
repair [47–49].
The artificial analogues of damages. An example of
artificial nucleotide injury is deoxyuridine or deoxy-
thymidine, containing the fluorescein residue, attached
to C5 position via the linker fragment (Fig. 5, a). The
monomer moiety for the synthesis of such DNA is a
commercially available amidite for the standard solid-
phase synthesis. It is also possible to introduce this mo-
dification using the DNA-polymerase reaction with the
modified dNTP as a substrate.
The category of artificial analogues of damages in-
cludes recently suggested derivatives of dCMP and
dUMP, containing the photoactivated arylazide groups,
linked to the nitrogenous bases via extended and flexi-
ble linker fragments (Fig. 5, b, c) [50]. It was demon-
strated that such arylazide derivatives of nucleotides in-
troduced into model DNA are recognized as substrates
by the bacterial NER system [51] as well as the NER sys-
tem of the mammalians [52]. The photoactivated ana-
logues of damages expand the capabilities of such re-
search instrument since they allow to perform the study
of a multicomponent system NER using the [53]. Du-
ring repair NER proteins form unstable specific comple-
xes of various compositions and architecture on the da-
maged DNA. The main idea of affinity modification ap-
proach is a covalent fixation of such protein complexes.
172
EVDOKIMOV A. N. ET AL.
O
O
O
HN
O
H
N
O
HN
N
O
O
O
O
P
O
O-
O
PO
O
O-
NH
O
O
NH
O
O
P
O
O-
O
PO
O
O-
NH
OHHO
O
O
O
O
NH
O
N
N
H
O
HN
N
N
O
F
N3
F Cl
H
N
N
N
H
OO
HN
N
F
N3
F Cl
H
N
O
a
b
c
d
e
Fig. 5. Synthetic analogous of damage: a – fluoresceine-5(6)-carboxy-
aminocaproyl-[5-(3-amino allyl)-2'-deoxyuridine; b – 5-{N-[N-(4-
azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-trans-3-
aminopropenyl-1}-2'-deoxyuridine; c – exo-N-[2-N-(N-(4-azido-2,5-
difluoro-3-chloropyridine-6-yl)-3-aminopropionyl)-aminoethyl]-2'-
deoxycytidine; d – non-nucleoside fragments of modified DNA
strand, containing N-[6-(9-antracenylcarbamoyl)hexanoyl]-3-amino-
1,2-propandiol; e – non-nucleoside fragments of modified DNA
strand, containing N-[6-(5(6)-fluoresceinylcarbamoyl)hexanoyl]-3-
amino-1,2-propandiol
Recently a novel artificial non-nucleoside damages
on the basis of N-[6-(9-antracenylcarbomoyl)hexanoyl]-
3-amino-1,2-propandiol (nAnt) and N-[6-(5(6)-fluo-
resceinylcarbomoyl)hexanoyl]-3-amino-1,2-propandi-
ol (nFlu) (Fig. 5, d, e) have been suggested, which imi-
tate NER-recognizable adducts [54].
The example of the synthetic analogues of the dama-
ges to be repaired by NER can be found in the oligopep-
tide-DNA adducts, the products of proteolytic degra-
dation of larger protein-DNA adducts, formed in the
cell under the impact of ionizing radiation, a number of
chemical reagents as well as the processes of DNA me-
tabolism [55]. The advantages of artificial damages are
the possibility of their introduction into the target posi-
tion of a DNA molecule and the possibility of varia-
tions in their structure, which is often required for the
experiments investigating the NER system in vitro.
The model DNA, used to investigate the NER
process. In order to form the functionally active prein-
cision NER complex, the damage should either be intro-
duced into a circular DNA [3], or into extended (� 120
bp) linear duplex [56].
The circular DNA with multiple damages. A number
of works, investigating the substrate properties of a mo-
del damages, have been performed using model struc-
tures created on the basis of phage or plasmid DNA,
which contain damages, statistically distributed along
the molecule. The damages are introduced into DNA
using either chemical modification or dosed UV-radia-
tion [57]. The subsequent product purification and its
analysis allow to estimate the amount and composition
of the introduced damages [58]. The characteristics of
the obtained model DNA depend on the type of the da-
maging reagent and the conditions, in which the treat-
ment of the DNA was performed. The context of DNA
sequence surrounding the target nucleobase is also im-
portant [59, 60]. The circular DNA, affected by high do-
ses of UV-radiation, was suggested as one of the first
model system for the research on the nucleotide exci-
sion repair. The irradiation of the plasmid DNA with
UV-light at the wavelength of 254 nm resulted in the
formation of both types of photoproducts. The obtained
substrate contained the mixture of cyclobutane pyrimi-
dine adducts and 6–4-photoproducts in random positi-
173
MODEL DNA FOR INVESTIGATION OF MECHANISM OF NUCLEOTIDE EXCISION REPAIR
a
elongation
ligation
b
introduction of
modified dNTPs
hybridization
with flanking
oligonucleotide
ligation
hybridization with
complementary
strand
c
ligation
hybridization with
complementary
strand
Fig. 6. Model DNA synthe-
sis methods: a – circle DNA;
b – synthesis of linear model
DNA using enzymatic ap-
proach; c – synthesis of li-
near model DNA using pre-
synthetized oligonucleotides
ons of DNA strands [58, 61]. There are evident prob-
lems and restrictions of such artificial NER substrates
use. Nevertheless, such model DNAs was applied to es-
timate the level of radioactive nucleotides incorpora-
tion during the reparative synthesis. This approach was
used for investigation of status of NER system [62, 63].
The circular DNA with the damage at the defined po-
sition of the strand. The fill-in method based on DNA-
polymerase-catalyzed reaction is widely used for the
synthesis of NER substrates, containing the damage (da-
mages) in the defined position (positions) of the DNA
strand. The modified oligonucleotide is used as a pri-
mer and the single-stranded form of the circular DNA
(plasmid or phage) is used as a template. Then the enzy-
matic ligation is used to restore the strand integrity
(Fig. 6, a) [64, 65]. The method of the modified primer
creation is defined by the type of the damage [19, 66–
68]. This approach to the synthesis of the NER system
substrates is rather labor-consuming and does not provi-
de a high yield, but it allows produce modified DNA of
certain structure and properties.
The circular DNAs with the damage in the defined
position were used to estimate the efficiency of the da-
mages elimination as well as to analyze the NER mecha-
nism [69]. The use of the circular DNA, containing the
regions of several uncoupled bases at the distance of 60
bp from the damage at 3'- or 5'-side, allowed confir-
ming the hypothesis of a bipartite mechanism of the da-
mage recognition. It was shown that the efficiency of re-
pair of model DNA, containing the cyclobutane pyrimi-
dine dimers that are hard to repair, increases conside-
rably with the introduction of 3nt bubble at 5'-side of the
damage. The further research, performed with recom-
binant dimer XPC-HR23B and factor TFIIH – the sys-
tem of recognition and control of the damage, reconsti-
tuted from the purified proteins – demonstrated that
connecting to the bubble, XPC promotes the assembly of
the protein complex, containing TFIIH. This complex
starts to melt the duplex and moves towards the 3'-side
from the initial binding place in the search for the da-
mage, containing the bulky chemical modification. Du-
ring this scanning the complex is translocated along the
DNA for the distance, measured with hundreds of base
pairs [70].
The linear DNA with the damage at the defined po-
sition of the strand. One of the most widely used inst-
ruments of in vitro NER investigation are linear dsDNA
with the damage at the defined position. Depending on
the suggested tasks, these model DNA may be of diffe-
rent length, structure and sequence.
The DNA-duplexes, not exceeding 50 bp, were used
to study the interaction of specific proteins of the NER
system with the damaged DNA by methods of affinity
modification, gel-retardation, equilibrium titration as well
as immunological techniques. Generally the obtaining
of these model DNAs does not cause any complications,
especially if the corresponding monomers were availab-
le for the automatic standard solid-phase synthesis.
The extended linear DNA-duplexes with the bulky
modification in the middle of the strand may be conside-
red as universal models, suitable for the experiments of
all the mentioned types [71–74]. Also such DNAs may
be used for the investigation of the excision of the da-
mage, as well as the changes of the nucleoprotein comp-
lexes, occurring during the NER process, which are as-
sociated with each stage [11, 75], including the ones in
the composition of artificial nucleosomes [76, 77].
These model DNAs may be also obtained via enzy-
matic ligation of the structure, composed of several
short overlapping oligonucleotide fragments. This ap-
proach is not complicated and allows varying the type
of the damage by replacing the modified oligonucleoti-
de, but the yield of the target extended DNA is not high –
5–6 % of the amount of oligonucleotide with the modifi-
cation [78].
The improved method, providing for considerably
higher yield, involves the ligation of the structure, com-
posed of the modified oligonucleotide, which is 12–16
nucleotides long, and two flanking rather extended (60–
70 nucleotides) oligonucleotides on the template of the
optimal length (30–35 nucleotides). The hybridization
of the obtained extended modified DNA and the comp-
lementary strand is performed to obtain the target dup-
lex (Fig. 6, c) [54].
Recently the method of creation of NER substrate
analogues, involving the use of several enzymes, inclu-
ding DNA-polymerases and DNA-ligase, has also be-
en elaborated (Fig. 6, b) [79]. A number of modified
dNTP, containing photoactive and fluorescent bulky sub-
stitutes introduced via long and flexible spacers along
the nitrogenous base, were used as substrates of DNA-
polymerase [50, 80, 81]. Good substrate properties of
174
EVDOKIMOV A. N. ET AL.
these dNTP and obtained modified oligonucleotides in
the reactions, catalyzed by the DNA-polymerase and
DNA-ligase, provided a high yield and wide variety of
the model DNAs for the NER system investigation [82].
The DNA-analogues of NER substrates as pro-
bes for the affinity modification. The method of pho-
toaffinity modification – covalent crosslinking of pro-
teins with DNA-substrate analogues, containing photo-
active groups – proved itself as informative approach to
the investigation of multicomponent nucleoprotein com-
plexes. The reaction of a covalent attachment may be
initiated via UV-irradiation at any suitable time after
the formation of a specific non-covalent protein-DNA
complex. The activation of photoreagents may be per-
formed in very short time. The adduction may be very
efficient in a wide pH range, ionic strength and the tem-
perature, whereas the reaction conditions often have to
be optimized for different types of reactive groups. UV-
induced adduction allows analyze even unstable comp-
lexes of variable composition. This is especially impor-
tant for the investigation on the multicomponent dyna-
mic systems of DNA metabolism including the NER
system. The variation of the structure of the model DNA
allows to investigate interaction between the NER pro-
tein factors and the DNA-substrate and their mutual im-
pact as well as the structure and mechanism of functio-
ning of the reparative complexes [51–53, 83]. The DNA
probes successfully used to study protein-DNA inter-
action contain the aryl azide group, introduced as a sub-
stitute into the pyrimidine base at C5 position of dU
(dT) or the exocyclic atom of nitrogen in dC [81]. After
UV-radiation the arylazide derivatives generate the short-
term singlet and long-term triplet nitrene, capable to ef-
ficiently react with a number of amino acid residues [80,
84, 85]. Being bulky substitutes, these photoreagents
distort the structure of the DNA duplex [86], while the
halogen- and tio-derivatives such as 5-J-dU and 4S-dU
(dT) introduce minimal changes into the DNA structu-
re [87]. Such derivatives do not provide high yield of
the covalent adducts, when the source of coherent radia-
tion of high radiation power (laser) was replaced with
more common sources of ultraviolet (high pressure mer-
cury lamp, etc.). However, the DNAs containing these
derivatives were applied for investigation of NER sys-
tem [88–91]. In addition, the reaction of p-azidophena-
cylbromide for the inter-nucleotide phosphorothioate
may be used to introduce photoactive groups into the
DNA-analogues of the NER substrates. The DNAs, con-
taining this modification, were used as photoactive ana-
logues of the undamaged chain [51]. The variation of the
type of reagents and damages and their mutual location
provides possibilities to design the efficient probes for
the NER the mechanism investigation using the affinity
modification method.
The experiments using the photoactive model DNA
resulted in a considerable advancement in the under-
standing of the mechanism of key stages of the NER eu-
karyotic system – damage recognition and preincision
complex formation. The pyrimidine nucleoside-5'-tri-
phosphates containing bulky fluorochloroazidobenzo-
yl (Fab) and fluorochloroazidopyridyl (Fap) groups ha-
ve good substrate properties in the DNA-polymerase re-
action and the efficiency as photoreagents. That allow-
ed to synthesize the set of DNA-probes with photoreac-
tive damages [50].
The analysis of the interaction of human recombi-
nant RPA and XPA with the modified DNA-duplexes of
different structure as well as single-stranded DNA, con-
taining Fab-dUMP (5-{[4-(4-azido-2,3,5,6-tetrafluoro-
benzoylamino) butyl]-aminocarbonyl-carbamoyl-pro-
pyl-oxymethyl}-2'-deoxyuridine-5'-monophosphate) or
fluorochloroazidopyridyl modifications Fap-dUMP (5-
{N-[N-(4-azido-2,5-difluoro-3-chloropyridyl-6-yl)-3-
aminopropionyl]-trans-3-aminopropenyl-1}-2'-deoxy-
uridine-5'-monophosphate) or Fap-dCMP (exo-N-{2-
[N-(4-azido-2,5-difluoro-3-chlorpyridine-6-yl)-3-ami-
nopropionyl]-aminoethyl}-2'-deoxycytidinine-5'-mono-
phosphate), demonstrated that the efficiency of photo-
affinity labeling depends on the structure of both DNA
and the photoanalogue. The presence of extended sing-
le-stranded fragments in the DNA-probe played a cru-
cial role in the interaction with RPA. The exception was
effective RPA-DNA adducts formation when the probe
represents DNA duplex containing nick flanked by Fab-
dNMP on 5'-side.
This was likely to be related to the substantial struc-
tural distortion in the DNA, induced by bulky modifi-
cation at the 5'-end of the oligonucleotide, bearing high
flexibility in the undamaged strand. XPA did not demon-
strate preference to single-stranded or double-stranded
DNA, and was interacted more efficiently with DNA-
duplexes containing nick [12].
175
MODEL DNA FOR INVESTIGATION OF MECHANISM OF NUCLEOTIDE EXCISION REPAIR
The interaction of XPC-HR23B, RPA and XPA with
the DNA-duplexes, containing the photoactive dama-
ges Fap-dC and Fap-dU in the inner positions, was also
studied. Fap-dC in the context of 3nt mismatch interac-
ted with XPC more efficiently. This is in agreement with
a higher level of specific excision of the damaged frag-
ment from the corresponding plasmid DNA. The level of
the photocrosslinking as well as the level of specific ex-
cision for Fap-dU did not depend considerably on the
complementarity of the pair, containing the damage [52].
To analyze NER factors capability to contact with
the damaged and undamaged strands of the substrate, the
set of DNA probes of different structure have been used.
Photoactive strand of the ds DNA contained damage-
imitating Fap-dNMP or not distorting 4-S-dUMP. The
second DNA strand either did not contain any modifica-
tions, or contained a bulky modified nucleotide (Antr-
dCMP) opposite the photoactive group. During the pho-
tocrosslinking of XPC-HR23B the covalent adducts with
48-bp-DNA probe were formed only by the XPC-subu-
nit of the heterodimer. The XPC modification level was
increased by the RPA presence. The amount of photo-
crosslinked XPC was below the detection level while
using the DNA with in oppositely located bulky modifi-
cations in both strands. This indicated that the binding
of XPC required the intact complementary strand oppo-
site the damage. The direct evidence of the role of the
undamaged DNA strand in this interaction was later ob-
tained using the X-ray structural analysis of the yeast or-
tholog of XPC complex and other experiments [92, 93].
The introduction of bulky modification into the second
strand decreased the level of RPA modification conside-
rably, which is also explained by preferential coupling
of the undamaged chain that is the function, performed
by RPA in the NER process [94, 95]. The presence of
the second damage did not impact the XPA modifica-
tion considerably. Therefore, the data, obtained due to
the experiments of the photocrosslinking of RPA and
XPA, indicates that the XPA factor prefer the damaged
chain, and the RPA factor – the undamaged chain [96].
The equilibrium fluorescent titration was used to es-
timate the parameters of the affinity of RPA to the seri-
es of DNA-duplexes, containing bulky substitutes, intro-
duced into the pyrimidine bases. Since this protein has
twice higher affinity to the single-stranded DNA than to
double-stranded DNA, it was assumed that in this case
RPA may serve as a specific sensor of a single-strand
character in the DNA-duplexes, caused by the distortion
of the DNA structure via the introduction of bulky sub-
stitutes. It was demonstrated that the affinity of the mo-
dified DNA exceeds 3–20-fold the affinity of this pro-
tein to the DNA-duplex, not containing any modifica-
tions. Using 5-J-dU-containing duplexes it was demonst-
rated that the level of RPA modification decreased ra-
pidly when an bulky substitute introdused in the comp-
lementary chain opposite photoactive nucleotide [79].
The stimulating effect of RPA on the formation of
XPC-HR23B complexes with DNA was observed in the
analysis of the interaction of proteins XPC-HR23B, XPA
and RPA with the duplexes, containing the photoreac-
tive residue 5-J-dUMP in one of the strands, and fluo-
rescein-replaced derivative of dUMP as the damage –
in the second one. The synergism in the interaction of
proteins XPA and XPC-HR23B with DNA was obser-
ved in case of their simultaneous presence in the reaction
mixture. The amount of modification products of pro-
teins XPA, RPA and XPC was depended on the mutual
location of the damage and the photoactivated group
and correlated with the affinity of these proteins to the
damaged DNA, estimated by the gel retardation [91].
The detailed analysis of the interaction of XPA and
RPA with the damaged and undamaged DNA strands
was performed using a series of photoactive J-dU-DNA
with different mutual location of the photoreagent and a
damaged nucleotide, which contains a fluorescein resi-
due [90]. For this experiments the model DNA mimi-
cking the repair intermediate (a DNA-duplex contai-
ning an noncomplementary fragment of 15 nucleoti-
des), were used. The application of the photocross-lin-
king methods, gel retardation and enzymatic footprin-
ting allowed determining the site of the damaged DNA,
with which these proteins contact more intensively. It
was confirmed that XPA and RPA mutually stimulate
their binding to DNA. It was also confirmed that RPA
mainly interacts with the undamaged DNA strand that
supported the conclusion that XPA has contacts with the
damaged chain, located at the 5'-side of the damage.
The asymmetry regarding the damaged and undamaged
chains and the synergism in the interaction of XPA and
RPA with the damaged DNA was also observed in some
previous works. The cisplatin-induced intrastrand lin-
king of DNA and cholesterol modifications were used
176
EVDOKIMOV A. N. ET AL.
as damages in these works, while J- and Br-dUMP as
well as J-dCMP were used as photoreagents [14, 88].
The photoaffinity labeling together with the estima-
tion of the affinity to the DNA-substrate by the gel retar-
dation and the equilibrium titration allowed comparing
the properties of human recombinant XPC-HR23B and
yeast Rad4/Rad23 [93]. The comparative analysis of the
interaction of two eukaryotic sensors with bulky dama-
ges and a set of model DNA-duplexes, containing a
fluorescein residue and 5-J-dUMP as a photoactive link
in different positions of the chain revealed crucial simi-
larity in their interaction with the damaged DNA and so-
me differences in the details of this interaction. The da-
ta on the contacts of these proteins with the DNA contai-
ning bulky lesion, supports model, created previously on
the basis of the results of the X-ray structural analysis.
In this analysis the crystals of the triple complex contai-
ning Rad23, the truncated form of Rad4 and the DNA-
duplex containing the UV-damage was used [92].
As for the duplexes, containing both Fap-dC and the
platinum adduct in one chain [82], regardless of the mu-
tual location of these bulky modifications in the DNA
molecule, the target of the modification was only a large
subunit of XPC-HR23B complex. The second adduct
with lower electrophoretic mobility is the most likely
product of photocrosslinking of the DNA-binding subu-
nit of XPC along other amino acid residues [89]. The
products of labeling of the 58 kDa regulatory subunit
HR23B were not detected in any reaction mixtures af-
ter the photocrosslinking. In addition, the product of
cross-linking between XPC and HR23B subunits, re-
vealed using the Western-blotting, was not have radio-
active label and is formed regardless of the presence of
the DNA-probe [82]. The absence of the products of
photocross-linking of HR23B to the analogues of the da-
maged DNA indicated that this subunit of the complex
does not participate in any direct contacts with DNA.
One might assume that the absence of the modification
products for a small subunit was conditioned by the to-
pology of the heterodimer coupling with DNA, steric
complications, caused by the location of subunits. How-
ever, it has been recently shown in the experiments in
vivo that a small subunit is released from the complex
while XPC binds to the damage [97].
To investigate the order of NER factors binding to
the DNA-substrate, the model DNA, containing the 1,3-
intrastrand link G*CG*, obtained in the reaction with
cis-diaminodichloroplatinum was used. It should be no-
ted that the structure of this model DNA is characteri-
zed in details. For instance, it is known that the axis of
dsDNA strand, containing 1,3-Pt-modification, forms
an approximate 150° angle, and the complementary in-
teractions of bases from the position «–3» (relative to
the damage) to the position «+3» were distorted [98–
100]. The photoaffinity modification experiments were
performed using DNA, containing the photoactive 5-
(N-p-azidobenzoyl)-3-aminoallyl)-dUMP (Az-dUMP)
in with such a Pt-damage in the same strand. The data,
obtained in the permanganate foot-printing in the pre-
sence of recombinant XPC-HR23B, RPA, XPA, XPF,
XPG and TFIIH (in different combinations), together
with the data of photocrosslinking allow to detect the
conformational changes in the damaged DNA. These
changes were induced by the binding of NER factors
and led to the formation of an asymmetric bubble around
the damage from the position «–20» to «+9». The order
of binding and positioning of these proteins was sug-
gested. However Az-dUMP can also introduce some
distortions in the dsDNA structure, the modification
patterns actually reflect the interaction of the NER fac-
tors and DNA, containing two damages, so the inter-
pretation of the photocross-linking results becomes
complicated.
The research, performed with the set of photoactive
model DNA, also provided insight into the mechanism
of functioning of the prokaryotic system NER (UvrABC).
One of the key stages of the work of the NER proka-
ryotic system, the mechanism of which has not been de-
termined for a long time, is the transfer of the damaged
DNA from UcrA to UvrB, preceding the UvrC-cata-
lyzed incision. Two types of arylazide photoaffinity re-
agents were used to determine this mechanism. The role
of the damage in the first type of probes was played by
the fluorescein residue, introduced as a substituent via a
linker on deoxytimidine. In this case p-azidophenacyl
derivative of deoxyoligonucleotide was used as a photo-
active analogue of the undamaged chain. Other probes
contained photoactive damages Fabc-dUMP or Fab-
dCMP, modified nucleotides, where a fluoroaryloben-
zoyl group was introduced via linkers on nitrogenous
bases. The application of photoactive DNA-analogues
of substrates revealed the principles of damage recog-
177
MODEL DNA FOR INVESTIGATION OF MECHANISM OF NUCLEOTIDE EXCISION REPAIR
nizion and the details of interaction between factors
UvrA, UrvB and UrvC and DNA in the NER process. It
was demonstrated that the «transfer» of the damaged
DNA from UvrA to UvrB consists of three stages. At
the first stage UvrA and UvrB are linked to the place of
the damage, and the direct contact with the damage is
performed by UvrA. At the second stage there is a reac-
tion of relocation of the damage containing fragment of
DNA strand with the ATP consumption, where UvrB
mainly contacts with the undamaged DNA, and at the
third stage a special pocket-like structure of UvrB binds
to the damage with the simultaneous release of UvrA.
Then the complex ready for the excision is formed with
the introduction of UvrC into this complex [51].
The photoaffinity modification technique was appli-
ed to search for mammalian cells NER-competent ex-
tracts proteins, specifically interacting with bulky sub-
stituted DNA. The proteins of HeLa cell extract selecti-
vely and efficiently interacted with Fap-dC- and Fap-
dU-duplexes (48 bp). The major targets of the modifica-
tion were several proteins with apparent molecular weight
of 35–90 kDa. The application of specific antibodies al-
lowed to identify a large RPA subunit (p70) as modifi-
cation target [89]. The experiments with the purified re-
combinant protein also demonstrated that RPA effici-
ently crosslinked with dsDNA, containing bulky dama-
ges. The major role in the interaction with damaged
DNA was played by p70 [79]. Using the more extended
DNA probe (137 bp Fap-dC-DNA) the relationship of
modification and the ratio of the concentrations of
DNA and protein extract was demonstrated. Decrease
of the concentration of extract proteins was led to the
broadening of the set of the targets forming covalent
adducts to DNA.
The most probable reason of the observed effect was
the increase in the possibility for proteins with the high-
er affinity to the damaged DNA to compete for the pho-
toactive group with the abundant protein of cancer cell
extracts – Ku70/80 antigen [101]. This assumption was
confirmed by the differences in the patterns of the
protein modification of the NER-competent extracts of
HeLa and the Chinese hamster ovarian cells (CHO).
The application of the functional test, based on the use
of NAD+, allowed identifying the 115-kDa protein of
the CHO extract, forming the adducts with the
extended DNA-probe, as PARP1 [102].
The experiments with dsDNA probes of different
length, containing photoactivated (benzophenone) deri-
vatives of cisplantin allowed performing the mass-
spectrometry identification of a number of proteins of
the mammalian cell extracts, interacting with the bulky
damage. It should be noted that there are differences in
the modification patterns, determined using the probes
of different length, containing similar modification. The
main targets of photocrosslinking for 1,3 Pt(GCG)-
DNA probe were Ku70/Ku80, DNA-dependent protein-
kinase, DNA-ligase III, PARP1 and RPA (p70) [73].
The model DNA and the estimation of the exci-
sion activity of the NER system. The recognition and
excision of damage is a key stage of NER. The accurate
and reliable evaluation of the specific excision activity
is required both for medical and fundamental research.
It would be useful for the investigation of the mecha-
nisms of bulky damage repair in reconstituted systems.
Also the estimation of the NER system activity in the tu-
mor cells would be useful to select appropriate chemo-
therapeutic drugs. It would probably help to correct the
method of anticancer treatment during therapy. Not very
sophisticated and convenient method of NER system
functional status control has not yet developed. One of
the important conditions for the development of this me-
thod is the existence of easily accessible and effective
substrate of excision. The long linear dsDNA appears
most promising for the elaboration of a NER activity
test system, despite some of the problems associated
with their use in the real systems like extracts of tissues
and cells [101, 103, 104]. The linear DNA substrates are
more available, especially if for their preparation can
be used the standard automatic solid-phase synthesis.
The second important characteristic is good sub-
strate properties of the model damage, high efficiency
of its conversion. For instance, the occurrence of the
products of the excision reaction was not registered for
the linear DNA duplex of 120 bp, containing the acetyl-
aminofluorene modification (AAF-dG). A well-de-
tectable level of excision of the AAF-dG-containing
fragment was observed only for the circular substrate
[27].
The model DNA, containing nAnt and nFlu have be-
en suggested as a substrates of NER system. The modi-
fied deoxyoligonucleotides were synthesized using the
standard solid-phase synthesis and the corresponding
178
EVDOKIMOV A. N. ET AL.
non-nucleosidic phosphoramidites. The nFlu and nAnt
containing extended (137 bp) linear DNA-duplexes de-
monstrated the properties of efficient substrates of spe-
cific excision reaction, catalyzed by the proteins of the
NER-competent extracts of eukaryotic cells. It has been
shown that incubation of these DNA with extracts of
CHO cells as well as the extracts, obtained from HeLa,
SiHa and C33A cells results in the formation of the frag-
ments of specific sequence and size. The levels of a rela-
tive activity of specific endonucleases of the extracts
differed 1.5–2-fold, and the excision profiles matched
for all the extracts. Therefore, the use of nFlu- and nAnt-
DNA allows estimating the level of the activity of the
NER system in the extracts of cells of different origin.
The radioactive labeling of the reaction products was
performed using the DNA-polymerase and �-32P-dCTP.
The template for fill-in synthesis was complementary
to the DNA fragment which contains the damage [105].
The 5'-end of the template contains the oligo-dG-frag-
ment; the 3'-end is protected from elongation with phos-
phorylation. This approach allows using the non-radio-
active model DNA. At the same time the sensitivity of
the method is considerably increased and one can reve-
al the products of the specific hydrolysis even at low ex-
cision levels.
Using the model DNA, containing the radioactive
label (32P) near the damage, it is possible to perform the
direct detection of the excision products [64]. The ad-
vantages of this method are the absence of the addi-
tional stages and the possibility of a quantitative estima-
tion of the excision efficiency. Considerable disadvan-
tages of this method should be also noted. Firstly, the
applied model DNA should have high relative radioac-
tivity which requires additional special protection du-
ring the synthesis and storage of these compounds. Se-
condly, such highly radioactive molecules have an ex-
tremely short lifetime which limits their application in
the series of experiments.
Conclusions. Using various methods including the
target mutagenesis, X-ray structural research as well as
fluorescent microscopy in the living cell, the conside-
rable progress have been done in the NER process in-
vestigation. The biochemical approaches based on the
application of the model DNA though remain very ap-
plicable to get insight of NER proteins interaction with
DNA-substrate and their mutual interactions in the spe-
cific nucleoprotein complexes [72, 106, 107]. The ac-
tuality of these investigations is also supported by the
broadening of the spectrum of the genotoxic factors. In
addition, the majority of antitumor agents, used in the
clinical practice, also realize their cytotoxicity via da-
maging DNA. However due to a wide substrate specifi-
city of NER, this repair system action interfere with the
safety of the damages, thus the medicinal effect of the
drugs is decreased.
Therefore, the understanding of the mechanisms of
functioning of protein complexes and the possibility of
estimation of the functional status of the NER system
using the model DNA are required for successful design
of drugs for the anticancer chemotherapy.
Funding. The work was supported by the Russian
Foundation for Fundamental Research (Grant 12-04-
00487a), the Russian Academy of Sciences (the pro-
grams of fundamental research in the field of molecular
and cell biology), the Ministry of Education and Sci-
ence of the Russian Federation (NSh-420.2014.4) and
COMFI 13-04-40197.
Ìîäåëüíûå ÄÍÊ äëÿ èññëåäîâàíèÿ ìåõàíèçìà íóêëåîòèäíîé
ýêñöèçèîííîé ðåïàðàöèè
À. Í. Åâäîêèìîâ, Î. È. Ëàâðèê, È. Î. Ïåòðóñåâà
Ðåçþìå
ÄÍÊ æèâûõ êëåòîê íàõîäèòñÿ ïîä ïîñòîÿííûì âîçäåéñòâèåì
ðàçëè÷íûõ ïîâðåæäàþùèõ ôàêòîðîâ ýêçî- è ýíäîãåííîãî ïðîèñ-
õîæäåíèÿ. Íóêëåîòèäíàÿ ýêñöèçèîííàÿ ðåïàðàöèÿ (NER) óäàëÿåò
èç ÄÍÊ øèðîêèé íàáîð îáúåìíûõ àääóêòîâ, îáðàçîâàâøèõñÿ â ðå-
çóëüòàòå âîçäåéñòâèÿ ÓÔ îáëó÷åíèÿ, à òàêæå ýëåêòðîôèëüíûõ
âåùåñòâ – çàãðÿçíèòåëåé îêðóæàþùåé ñðåäû, îêàçûâàþùèõ ìó-
òàãåííîå äåéñòâèå, è õèìèîïðåïàðàòîâ. Â ïðîöåññå ðåïàðàöèè,
ïðîâîäèìîé ñèñòåìîé NER ìëåêîïèòàþùèõ, ïðîèñõîäèò ñïåöè-
ôè÷åñêîå âûùåïëåíèå èç ÄÍÊ ôðàãìåíòîâ ðàçìåðîì 24–32 íóêëåî-
òèäà, ñîäåðæàùèõ ïîâðåæäåíèÿ. Ïîñëåäóþùèé ðåïàðàòèâíûé
ñèíòåç è ëèãèðîâàíèå ÄÍÊ âîññòàíàâëèâàþò èíòàêòíîñòü ñïè-
ðàëè ÄÍÊ. Èäåíòèôèöèðîâàíû ãåíû, èíàêòèâèðîâàííûå â NER-
äåôèöèòíûõ êëåòêàõ âûñøèõ ýóêàðèîòîâ.  ðåïàðàöèè ó÷àñòâó-
þò ïðèìåðíî 30 áåëêîâ, ôîðìèðóþùèõ ñïåöèôè÷åñêèå ìíîãîñóáú-
åäèíè÷íûå êîìïëåêñû. Ñèñòåìà NER õàðàêòåðèçóåòñÿ øèðîêîé
ñóáñòðàòíîé ñïåöèôè÷íîñòüþ è ïðè ýòîì áîëüøèìè ðàçëè÷èÿìè
â ýôôåêòèâíîñòè óäàëåíèÿ ïîâðåæäåíèé. Êëþ÷åâîé ëèìèòèðóþ-
ùåé ñòàäèåé ïðîöåññà ÿâëÿåòñÿ óçíàâàíèå è âåðèôèêàöèÿ ïîâðåæ-
äåíèÿ. Ê ýôôåêòèâíûì è ðàçâèâàþùèìñÿ ïîäõîäàì ê èññëåäîâà-
íèþ ïðîöåññà NER ïðèíàäëåæèò ìåòîä, îñíîâàííûé íà èñïîëü-
çîâàíèè ìîäåëüíûõ ÄÍÊ – ñèíòåòè÷åñêèõ ñòðóêòóð, ÿâëÿþùèõ-
ñÿ àíàëîãàìè ñóáñòðàòà èëè èíòåðìåäèàòîâ ýòîãî ïðîöåññà.
Ðàññìîòðåíû ñóùåñòâóþùèå äàííûå î ñïîñîáàõ êîíñòðóèðîâà-
íèÿ ìîäåëüíûõ ÄÍÊ è ïðèìåíåíèè èõ â êà÷åñòâå èíñòðóìåíòà äëÿ
âñåñòîðîííåãî èçó÷åíèÿ ïðîöåññà NER.
Êëþ÷åâûå ñëîâà: íóêëåîòèäíàÿ ýêñöèçèîííàÿ ðåïàðàöèÿ, îáú-
åìíûå ÄÍÊ-àääóêòû, ìîäåëüíûå ÄÍÊ.
179
MODEL DNA FOR INVESTIGATION OF MECHANISM OF NUCLEOTIDE EXCISION REPAIR
Ìîäåëüí³ ÄÍÊ äëÿ äîñë³äæåííÿ ìåõàíèçìó íóêëåîòèäíî¿
åêñöèç³éíî¿ ðåïàðàö³¿
À. Í. ªâäîêèìîâ, Î. ². Ëàâðèê, ². Î. Ïåòðóñåâà
Ðåçþìå
ÄÍÊ æèâèõ êë³òèí ïåðåáóâຠï³ä ïîñò³éíèì âïëèâîì ð³çíî-
ìàí³òíèõ ïîøêîäæóþ÷èõ ôàêòîð³â åêçî- ³ åíäîãåííîãî ïîõîä-
æåííÿ. Íóêëåîòèäíà åêñöèç³éíà ðåïàðàö³ÿ (NER) âèäàëÿº ç ÄÍÊ
øèðîêèé íàá³ð îá’ºìíèõ àäóêò³â, ÿê³ óòâîðèëèñÿ â ðåçóëüòàò³ 䳿
ÓÔ îïðîì³íåííÿ, à òàêîæ åëåêòðîô³ëüíèõ ðå÷îâèí – çàáðóäíþâà-
÷³â äîâê³ëëÿ, ùî ÷èíÿòü ìóòàãåííèé âïëèâ, òà õ³ì³îïðåïàðàò³â. Ó
ïðîöåñ³ ðåïàðàö³¿, ÿêó âèêîíóº ñèñòåìà NER ññàâö³â, â³äáóâàºòü-
ñÿ ñïåöèô³÷íå âèùåïëþâàííÿ ç ÄÍÊ ôðàãìåíò³â ðîçì³ðîì 24–32
íóêëåîòèäè, ùî ì³ñòÿòü ïîøêîäæåííÿ. Ïîäàëüøèé ðåïàðàòèâ-
íèé ñèíòåç ³ ë³ãóâàííÿ ÄÍÊ â³äíîâëþþòü ³íòàêòí³ñòü ñï³ðàë³ ÄÍÊ.
²äåíòèô³êîâàíî ãåíè, ³íàêòèâîâàí³ â NER-äåô³öèòíèõ êë³òèíàõ
âèùèõ åâêàð³îò³â.  ðåïàðàö³¿ áåðóòü ó÷àñòü ïðèáëèçíî 30 á³ëê³â,
ÿê³ ôîðìóþòü ñïåöèô³÷í³ ìóëüòèñóáîäèíè÷í³ êîìïëåêñè. Ñèñòå-
ìà NER õàðàêòåðèçóºòüñÿ øèðîêîþ ñóáñòðàòíîþ ñïåöèôè÷í³-
ñòþ ³ ïðè öüîìó âåëèêèìè ðîçá³æíîñòÿìè â åôåêòèâíîñò³ âèäà-
ëåííÿ ïîøêîäæåíü. Êëþ÷îâîþ ë³ì³òóþ÷îþ ñòà䳺þ ïðîöåñó º óï³-
çíàâàííÿ òà âåðèô³êàö³ÿ ïîøêîäæåí. Äî åôåêòèâíèõ ³ òàêèõ, ùî
ðîçâèâàþòüñÿ, ï³äõîä³â äî âèâ÷åííÿ ïðîöåñó NER íàëåæèòü ìå-
òîä, çàñíîâàíèé íà âèêîðèñòàíí³ ìîäåëüíèõ ÄÍÊ – ñèíòåòè÷íèõ
ñòðóêòóð, ÿê³ º àíàëîãàìè ñóáñòðàòà àáî ³íòåðìåä³àò³â öüîãî
ïðîöåñó. Ðîçãëÿíóòî ³ñíóþ÷³ äàí³ ùîäî ñïîñîá³â êîíñòðóþâàííÿ
ìîäåëüíèõ ÄÍÊ òà çàñòîñóâàííÿ ¿õ ÿê ³íñòðóìåíòà äëÿ âñåá³÷íî-
ãî äîñë³äæåííÿ ïðîöåñó NER.
Êëþ÷îâ³ ñëîâà: íóêëåîòèäíà åêñöèç³éíà ðåïàðàö³ÿ, îá’ºìí³
ÄÍÊ-àäóêòè, ìîäåëüí³ ÄÍÊ.
REFERENCES
1. Dip R, Camenisch U, Naegeli H. Mechanisms of DNA damage
recognition and strand discrimination in human nucleotide ex-
cision repair. DNA Repair (Amst). 2004; 3(11):1409–23.
2. Scharer OD. Chemistry and biology of DNA repair. Angew
Chem Int Ed Engl. 2003; 42(26):2946–74.
3. Gillet LC, Scharer OD. Molecular mechanisms of mammalian
global genome nucleotide excision repair. Chem Rev. 2006; 106
(2):253–76.
4. Lehmann AR. DNA repair-deficient diseases, xeroderma pig-
mentosum, Cockayne syndrome and trichothiodystrophy. Bio-
chimie. 2003; 85(11):1101–11.
5. Hanawalt PC, Spivak G. Transcription-coupled DNA repair:
two decades of progress and surprises. Nat Rev Mol Cell Biol.
2008; 9(12):958–70.
6. Mellon I, Spivak G, Hanawalt PC. Selective removal of trans-
cription-blocking DNA damage from the transcribed strand of the
mammalian DHFR gene. Cell. 1987; 51(2):241–9.
7. Scharer OD. Nucleotide excision repair in eukaryotes. Cold
Spring Harb Perspect Biol. 2003; 5(10):a012609.
8. Friedberg EC. How nucleotide excision repair protects against
cancer. Nat Rev Cancer. 2001; 1(1):22–33.
9. Sugasawa K, Okamoto T, Shimizu Y, Masutani C, Iwai S, Hanao-
ka F. A multistep damage recognition mechanism for global ge-
nomic nucleotide excision repair. Genes Dev. 2001; 15(5):507–21.
10. Kusumoto R, Masutani C, Sugasawa K, Iwai S, Araki M, Uchida
A, Mizukoshi T, Hanaoka F. Diversity of the damage recogni-
tion step in the global genomic nucleotide excision repair in vitro.
Mutat Res. 2001; 485(3):219–27.
11. Geacintov NE, Broyde S, Buterin T, Naegeli H, Wu M, Yan S, Pa-
tel DJ. Thermodynamic and structural factors in the removal of
bulky DNA adducts by the nucleotide excision repair machine-
ry. Biopolymers. 2002; 65(3):202–10.
12. Maltseva EA, Rechkunova NI, Petruseva IO, Silnikov VN, Ver-
meulen W, Lavrik OI. Interaction of nucleotide excision repair
factors RPA and XPA with DNA containing bulky photoreac-
tive groups imitating damages. Biochemistry (Mosc). 2006; 71
(3):270–8.
13. Tapias A, Auriol J, Forget D, Enzlin JH, Scharer OD, Coin F,
Coulombe B, Egly JM. Ordered conformational changes in da-
maged DNA induced by nucleotide excision repair factors. J
Biol Chem. 2004; 279(18):19074–83.
14. Hermanson-Miller IL, Turchi JJ. Strand-specific binding of RPA
and XPA to damaged duplex DNA. Biochemistry. 2002; 41 (7):
2402–8.
15. Sancar A. Structure and function of DNA photolyase and crypto-
chrome blue-light photoreceptors. Chem Rev. 2003; 103(6):
2203–37.
16. Reardon JT, Sancar A. Recognition and repair of the cyclobu-
tane thymine dimer, a major cause of skin cancers, by the human
excision nuclease. Genes Dev. 2003; 17(20):2539–51.
17. Mitchell DL. The relative cytotoxicity of (6-4) photoproducts
and cyclobutane dimers in mammalian cells. Photochem Pho-
tobiol. 1988; 48(1):51–7.
18. Smith CA, Taylor JS. Preparation and characterization of a set of
deoxyoligonucleotide 49-mers containing site-specific cis-syn,
trans-syn-I, (6-4), and Dewar photoproducts of thymidylyl
(3'�5')-thymidine. J Biol Chem. 1993; 268(15):11143–51.
19. Szymkowski DE, Lawrence CW, Wood RD. Repair by human
cell extracts of single (6-4) and cyclobutane thymine-thymine
photoproducts in DNA. Proc Natl Acad Sci USA. 1993; 90(21):
9823–7.
20. Mocquet V, Kropachev K, Kolbanovskiy M, Kolbanovskiy A, Ta-
pias A, Cai Y, Broyde S, Geacintov NE, Egly JM. The human DNA
repair factor XPC-HR23B distinguishes stereoisomeric benzo-
[a]pyrenyl-DNA lesions. EMBO J. 2007; 26(12):2923–32.
21. Reeves DA, Mu H, Kropachev K, Cai Y, Ding S, Kolbanovskiy A,
Kolbanovskiy M, Chen Y, Krzeminski J, Amin S, Patel DJ, Broy-
de S, Geacintov NE. Resistance of bulky DNA lesions to nucleo-
tide excision repair can result from extensive aromatic lesion-
base stacking interactions. Nucleic Acids Res. 2011; 39(20):
8752–64.
22. Kropachev K, Kolbanovskii M, Cai Y, Rodriguez F, Kolbanovs-
kii A, Liu Y, Zhang L, Amin S, Patel D, Broyde S, Geacintov NE.
The sequence dependence of human nucleotide excision repair
efficiencies of benzo[a]pyrene-derived DNA lesions: insights
into the structural factors that favor dual incisions. J Mol Biol.
2009; 386(5):1193–203.
23. Cai Y, Geacintov NE, Broyde S. Nucleotide excision repair effi-
ciencies of bulky carcinogen-DNA adducts are governed by a ba-
lance between stabilizing and destabilizing interactions. Bioche-
mistry. 2012; 51(7):1486–99.
24. Kropachev K, Kolbanovskiy M, Liu Z, Cai Y, Zhang L, Schwaid
AG, Kolbanovskiy A, Ding S, Amin S, Broyde S, Geacintov NE.
Adenine-DNA adducts derived from the highly tumorigenic Di-
benzo[a,l]pyrene are resistant to nucleotide excision repair whi-
le guanine adducts are not. Chem Res Toxicol. 2013; 26(5):
783–93.
25. Baertschi SW, Raney KD, Shimada T, Harris TM, Guengerich
FP. Comparison of rates of enzymatic oxidation of aflatoxin B1,
aflatoxin G1, and sterigmatocystin and activities of the epoxides
in forming guanyl-N7 adducts and inducing different genetic res-
ponses. Chem Res Toxicol. 1989; 2(2):114–2.
180
EVDOKIMOV A. N. ET AL.
26. Gan J, Skipper PL, Gago-Dominguez M, Arakawa K, Ross RK,
YuMC, Tannenbaum SR. Alkylaniline-hemoglobin adducts and
risk of non-smoking-related bladder cancer. J Natl Cancer Inst.
2004; 96(19):1425–31.
27. Gillet LC, Alzeer J, Scharer OD. Site-specific incorporation of
N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into
oligonucleotides using modified «ultra-mild» DNA synthesis.
Nucleic Acids Res. 2005; 33(6):1961–9.
28. Johnson DL, Reid TM, Lee MS, King CM, Romano LJ. Prepa-
ration and characterization of a viral DNA molecule containing a
site-specific 2-aminofluorene adduct: a new probe for mutagene-
sis by carcinogens. Biochemistry. 1986; 25(2):449–56.
29. O’Handley SF, Sanford DG, Xu R, Lester CC, Hingerty BE,
Broyde S, Krugh TR. Structural characterization of an N-acetyl-
2-aminofluorene (AAF) modified DNA oligomer by NMR, ener-
gy minimization, and molecular dynamics. Biochemistry. 1993;
32(10):2481–97.
30. Alzeer J, Scharer OD. A modified thymine for the synthesis of si-
te-specific thymine-guanine DNA interstrand crosslinks. Nucleic
Acids Res. 2006; 34(16):4458–66.
31. el-Bayoumy K, Chae YH, Upadhyaya P, Rivenson A, Kurtzke C,
Reddy B, Hecht SS. Comparative tumorigenicity of benzo[a]py-
rene, 1-nitropyrene and 2-amino-1-methyl-6-phenylimidazo[4,
5-b]pyridine administered by gavage to female CD rats. Carcino-
genesis. 1995; 16(2):431–4.
32. Delclos KB, Miller DW, Lay JO, Jr., Casciano DA, Walker RP,
Fu PP, Kadlubar FF. Identification of C8-modified deoxyino-
sine and N2- and C8-modified deoxyguanosine as major pro-
ducts of the in vitro reaction of N-hydroxy-6-aminochrysene
with DNA and the formation of these adducts in isolated rat he-
patocytes treated with 6-nitrochrysene and 6-aminochrysene.
Carcinogenesis. 1987; 8(11):1703–9.
33. Chae YH, Delclos KB, Blaydes B, el-Bayoumy K. Metabolism
and DNA binding of the environmental colon carcinogen 6-nitro-
chrysene in rats. Cancer Res. 1996; 56(9):2052–8.
34. Brooks PJ, Wise DS, Berry DA, Kosmoski JV, Smerdon MJ, So-
mers RL, Mackie H, Spoonde AY, Ackerman EJ, Coleman K, Ta-
rone RE, Robbins JH. The oxidative DNA lesion 8,5'-(S)-
cyclo-2'-deoxyadenosine is repaired by the nucleotide excision
repair pathway and blocks gene expression in mammalian cells.
J Biol Chem. 2000; 275(29):22355–62.
35. El-Bayoumy K, Sharma AK, Lin JM, Krzeminski J, Boyiri T, King
LC, Lambert G, Padgett W, Nesnow S, Amin S. Identification of
5-(deoxyguanosin-N2-yl)-1,2-dihydroxy-1,2-dihydro-6-amino
chrysene as the major DNA lesion in the mammary gland of rats
treated with the environmental pollutant 6-nitrochrysene. Chem
Res Toxicol. 2004; 17(12):1591–9.
36. Krzeminski J, Kropachev K, Kolbanovskiy M, Reeves D, Kolba-
novskiy A, Yun BH, Geacintov NE, Amin S, El-Bayoumy K. In-
efficient nucleotide excision repair in human cell extracts of the
N-(deoxyguanosin-8-yl)-6-aminochrysene and 5-(deoxyguano-
sin-N(2)-yl)-6-aminochrysene adducts derived from 6-nitrochry-
sene. Chem Res Toxicol. 2011; 24(1):65–72.
37. Zamble DB, Mu D, Reardon JT, Sancar A, Lippard SJ. Repair of
cisplatin-DNA adducts by the mammalian excision nuclease.
Biochemistry. 1996; 35(31):10004–13.
38. De Silva IU, McHugh PJ, Clingen PH, Hartley JA. Defining the
roles of nucleotide excision repair and recombination in the re-
pair of DNA interstrand cross-links in mammalian cells. Mol
Cell Biol. 2000; 20(21):7980–90.
39. Johnson NP, Macquet JP, Wiebers JL, Monsarrat B. Structures
of the adducts formed between [Pt(dien)Cl]Cl and DNA in vitro.
Nucleic Acids Res. 1982; 10(17):5255–71.
40. Jamieson ER, Lippard SJ. Structure, recognition, and processing
of cisplatin-DNA adducts. Chem Rev. 1999; 99(9):2467–98.
41. Kasparkova J, Novakova O, Farrell N, Brabec V. DNA binding
by antitumor trans-[PtCl2(NH3)(thiazole)]. Protein recognition
and nucleotide excision repair of monofunctional adducts. Bio-
chemistry. 2003; 42(3):792–800.
42. Wang D, Zhu G, Huang X, Lippard SJ. X-ray structure and me-
chanism of RNA polymerase II stalled at an antineoplastic mono-
functional platinum-DNA adduct. Proc Natl Acad Sci USA.
2010; 107(21):9584–9.
43. Zhu G, Myint M, Ang WH, Song L, Lippard SJ. Monofunctional
platinum-DNA adducts are strong inhibitors of transcription and
substrates for nucleotide excision repair in live mammalian cells.
Cancer Res. 2012; 72(3):790–800.
44. Park GY, Wilson JJ, Song Y, Lippard SJ. Phenanthriplatin, a mo-
nofunctional DNA-binding platinum anticancer drug candidate
with unusual potency and cellular activity profile. Proc Natl
Acad Sci USA. 2012; 109(30):11987–92.
45. Chen CH, Dickman KG, Moriya M, Zavadil J, Sidorenko VS,
Edwards KL, Gnatenko DV, Wu L, Turesky RJ, Wu XR, Pu YS,
Grollman AP. Aristolochic acid-associated urothelial cancer in
Taiwan. Proc Natl Acad Sci USA. 2012; 109(21):8241–6.
46. Grollman AP, Shibutani S, Moriya M, Miller F, Wu L, Moll U,
Suzuki N, Fernandes A, Rosenquist T, Medverec Z, Jakovina K,
Brdar B, Slade N, Turesky RJ, Goodenough AK, Rieger R, Vuke-
lic M, Jelakovic B. Aristolochic acid and the etiology of endemic
(Balkan) nephropathy. Proc Natl Acad Sci USA. 2007; 104(29):
12129–34.
47. Zhang F, Chen Y, Pisha E, Shen L, Xiong Y, van Breemen RB,
Bolton JL. The major metabolite of equilin, 4-hydroxyequilin,
autoxidizes to an o-quinone which isomerizes to the potent cyto-
toxin 4-hydroxyequilenin-o-quinone. Chem Res Toxicol. 1999;
12(2):204–13.
48. Zhang N, Ding S, Kolbanovskiy A, Shastry A, Kuzmin VA, Bolton
JL, Patel DJ, Broyde S, Geacintov NE. NMR and computational
studies of stereoisomeric equine estrogen-derived DNA cytidi-
ne adducts in oligonucleotide duplexes: opposite orientations of
diastereomeric forms. Biochemistry. 2009; 48(30):7098–109.
49. Okahashi Y, Iwamoto T, Suzuki N, Shibutani S, Sugiura S, Itoh S,
Nishiwaki T, Ueno S, Mori T. Quantitative detection of 4-hydro-
xyequilenin-DNA adducts in mammalian cells using an immuno-
assay with a novel monoclonal antibody. Nucleic Acids Res.
2010; 38(12):e133.
50. Dezhurov SV, Khodyreva SN, Plekhanova ES, Lavrik OI. A new
highly efficient photoreactive analogue of dCTP. Synthesis, cha-
racterization, and application in photoaffinity modification of
DNA binding proteins. Bioconjug Chem. 2005; 16(1):215–22.
51. DellaVecchia MJ, Croteau DL, Skorvaga M, Dezhurov SV, Lav-
rik OI, Van Houten B. Analyzing the handoff of DNA from
UvrA to UvrB utilizing DNA-protein photoaffinity labeling. J
Biol Chem. 2004; 279(43):45245–56.
52. Maltseva EA, Rechkunova NI, Gillet LC, Petruseva IO, Scharer
OD, Lavrik OI. Crosslinking of the NER damage recognition
proteins XPC-HR23B, XPA and RPA to photoreactive probes
that mimic DNA damages. Biochim Biophys Acta. 2007; 1770
(5):781–9.
53. Rechkunova NI, Lavrik OI. Nucleotide excision repair in higher
eukaryotes: mechanism of primary damage recognition in global
genome repair. Subcell Biochem. 2010; 50:251–77.
54. Evdokimov A, Petruseva I, Tsidulko A, Koroleva L, Serpokrylo-
va I, Silnikov V, Lavrik O. New synthetic substrates of mamma-
lian nucleotide excision repair system. Nucleic Acids Res. 2013;
41(12):e123.
181
MODEL DNA FOR INVESTIGATION OF MECHANISM OF NUCLEOTIDE EXCISION REPAIR
55. Reardon JT, Sancar A. Repair of DNA-polypeptide crosslinks by
human excision nuclease. Proc Natl Acad Sci USA. 2006; 103
(11):4056–61.
56. Huang JC, Sancar A. Determination of minimum substrate size
for human excinuclease. J Biol Chem. 1994; 269(29):19034–40.
57. Biggerstaff M, Robins P, Coverley D, Wood RD. Effect of exo-
genous DNA fragments on human cell extract-mediated DNA re-
pair synthesis. Mutat Res. 1991; 254(3):217–24.
58. Buterin T, Hess MT, Gunz D, Geacintov NE, Mullenders LH,
Naegeli H. Trapping of DNA nucleotide excision repair factors
by nonrepairable carcinogen adducts. Cancer Res. 2002; 62
(15):4229–35.
59. Hansson J, Wood RD. Repair synthesis by human cell extracts in
DNA damaged by cis- and trans-diamminedichloroplatinum
(II). Nucleic Acids Res. 1989; 17(20):8073–91.
60. Heiger-Bernays WJ, Essigmann JM, Lippard SJ. Effect of the an-
titumor drug cis-diamminedichloroplatinum(II) and related plati-
num complexes on eukaryotic DNA replication. Biochemistry.
1990; 29(36):8461–6.
61. Hess MT, Gunz D, Naegeli H. A repair competition assay to as-
sess recognition by human nucleotide excision repair. Nucleic
Acids Res. 1996; 24(5):824–8.
62. Sibghatullah, Husain I, Carlton W, Sancar A. Human nucleotide
excision repair in vitro: repair of pyrimidine dimers, psoralen
and cisplatin adducts by HeLa cell-free extract. Nucleic Acids
Res. 1989; 17(12):4471–84.
63. Araki M, Masutani C, Takemura M, Uchida A, Sugasawa K,
Kondoh J, Ohkuma Y, Hanaoka F. Centrosome protein centrin
2/caltractin 1 is part of the xeroderma pigmentosum group C
complex that initiates global genome nucleotide excision repair.
J Biol Chem. 2001; 276(22):18665–72.
64. Huang JC, Svoboda DL, Reardon JT, Sancar A. Human nucleo-
tide excision nuclease removes thymine dimers from DNA by in-
cising the 22
nd
phosphodiester bond 5' and the 6
th
phosphodiester
bond 3' to the photodimer. Proc Natl Acad Sci USA. 1992; 89
(8):3664–8.
65. Svoboda DL, Taylor JS, Hearst JE, Sancar A. DNA repair by eu-
karyotic nucleotide excision nuclease. Removal of thymine di-
mer and psoralen monoadduct by HeLa cell-free extract and of
thymine dimer by Xenopus laevis oocytes. J Biol Chem. 1993;
268(3):1931–6.
66. Szymkowski DE, Yarema K, Essigmann JM, Lippard SJ, Wood
RD. An intrastrand d(GpG) platinum crosslink in duplex M13
DNA is refractory to repair by human cell extracts. Proc Natl
Acad Sci USA. 1992; 89(22):10772–6.
67. Hansson J, Munn M, Rupp WD, Kahn R, Wood RD. Localization
of DNA repair synthesis by human cell extracts to a short region
at the site of a lesion. J Biol Chem. 1989; 264(36):21788–92.
68. Koehl P, Burnouf D, Fuchs RP. Construction of plasmids contai-
ning a unique acetylaminofluorene adduct located within a muta-
tion hot spot. A new probe for frameshift mutagenesis. J Mol
Biol. 1989; 207(2):355–64.
69. Thorel F, Constantinou A, Dunand-Sauthier I, Nouspikel T, Lal-
le P, Raams A, Jaspers NG, Vermeulen W, Shivji MK, Wood RD,
Clarkson SG. Definition of a short region of XPG necessary for
TFIIH interaction and stable recruitment to sites of UV damage.
Mol Cell Biol. 2004; 24(24):10670–80.
70. Sugasawa K, Akagi J, Nishi R, Iwai S, Hanaoka F. Two-step re-
cognition of DNA damage for mammalian nucleotide excision
repair: Directional binding of the XPC complex and DNA strand
scanning. Mol Cell. 2009; 36(4):642–53.
71. Roche Y, Zhang D, Segers-Nolten GM, Vermeulen W, Wyman C,
Sugasawa K, Hoeijmakers J, Otto C. Fluorescence correlation
spectroscopy of the binding of nucleotide excision repair protein
XPC-hHr23B with DNA substrates. J Fluoresc. 2008; 18(5):
987–95.
72. Yeo JE, Khoo A, Fagbemi AF, Scharer OD. The efficiencies of
damage recognition and excision correlate with duplex destabili-
zation induced by acetylaminofluorene adducts in human nucleo-
tide excision repair. Chem Res Toxicol. 2012; 25(11):2462–8.
73. Guggenheim ER, Xu D, Zhang CX, Chang PV, Lippard SJ. Pho-
toaffinity isolation and identification of proteins in cancer cell
extracts that bind to platinum-modified DNA. Chembiochem.
2009; 10(1):141–57.
74. Evdokimov AN, Tsidulko AYu, Petruseva IO, Koroleva LS, Ser-
pokrylova IYu, Silnikov VN, Lavrik OI. Synthesis of model DNA
and their application as substrates of nucleotide excision repair.
Biopolym. Cell. 2012; 28(3):212–7.
75. Hess MT, Gunz D, Luneva N, Geacintov NE, Naegeli H. Base pair
conformation-dependent excision of benzo[a]pyrene diol epoxi-
de-guanine adducts by human nucleotide excision repair enzymes.
Mol Cell Biol. 1997; 17(12):7069–76.
76. Todd RC, Lippard SJ. Consequences of cisplatin binding on nuc-
leosome structure and dynamics. Chem Biol. 2010; 17(12):
1334–43.
77. Fei J, Kaczmarek N, Luch A, Glas A, Carell T, Naegeli H. Regu-
lation of nucleotide excision repair by UV-DDB: prioritization of
damage recognition to internucleosomal DNA. PLoS Biol. 2011;
9(10):e1001183.
78. Huang JC, Hsu DS, Kazantsev A, Sancar A. Substrate spectrum
of human excinuclease: repair of abasic sites, methylated bases,
mismatches and bulky adducts. Proc Natl Acad Sci USA. 1994;
91(25):12213–17.
79. Petruseva IO, Tikhanovich IS, Chelobanov BP, Lavrik OI. RPA
repair recognition of DNA containing pyrimidines bearing bulky
adducts. J Mol Recognit. 2008; 21(3):154–62.
80. Wlassoff WA, Dobrikov MI, Safronov IV, Dudko RY, Bogachev
VS, Kandaurova VV, Shishkin GV, Dymshits GM, Lavrik OI. Syn-
thesis and characterization of (d)NTP derivatives substituted with
residues of different photoreagents. Bioconjug Chem. 1995; 6
(4):352–60.
81. Kolpashchikov DM, Zakharenko AL, Dezhurov SV, Rechkunova
NI, Khodyreva SN, Degtiarev S, Litvak VV, Lavrik OI. New re-
agents for affinity modification of biopolymers. Photoaffinity
modification of Tte-DNA polymerase. Bioorg Khim. 1999; 25
(2):129–36.
82. Neher TM, Rechkunova NI, Lavrik OI, Turchi JJ. Photo-cross-
linking of XPC-Rad23B to cisplatin-damaged DNA reveals con-
tacts with both strands of the DNA duplex and spans the DNA
adduct. Biochemistry. 2010; 49(4):669–78.
83. Khodyreva SN, Lavrik OI. Photoaffinity labeling technique for
studying DNA replication and DNA repair. Curr Med Chem.
2005; 12(6):641–55.
84. Knorre DG, Godovikova TS. Photoaffinity labeling as an approach
to study supramolecular nucleoprotein complexes. FEBS Lett.
1998; 433(1–2):9–14.
85. Zakharenko AL, Khodyreva SN, Rechkunova NI, Safronov IV,
Pyshnyi DV, Degtyarev SK, Lavrik OI. Photoaffinity labeling of
DNA polymerase from Thermus thermophilus and DNA templa-
te by photoreactive analogs of dCTP. Biochemistry (Mosc).
1998; 63(8):929–34.
86. Nguyen HK, Auffray P, Asseline U, Dupret D, Thuong NT. Modi-
fication of DNA duplexes to smooth their thermal stability inde-
pendently of their base content for DNA sequencing by hybri-
dization. Nucleic Acids Res. 1997; 25(15):3059–65.
87. Meisenheimer KM, Koch TH. Photocross-linking of nucleic
acids to associated proteins. Crit Rev Biochem Mol Biol. 1997;
32(2):101–40.
182
EVDOKIMOV A. N. ET AL.
88. Schweizer U, Hey T, Lipps G, Krauss G. Photocrosslinking lo-
cates a binding site for the large subunit of human replication
protein A to the damaged strand of cisplatin-modified DNA.
Nucleic Acids Res. 1999; 27(15):3183–9.
89. Petruseva IO, Tikhanovich IS, Maltseva EA, Safronov IV, Lav-
rik OI. Photoactivated DNA analogs of substrates of the nucleo-
tide excision repair system and their interaction with proteins of
NER-competent HeLa cell extract. Biochemistry (Mosc). 2009;
74(5):491–501.
90. Krasikova YS, Rechkunova NI, Maltseva EA, Petruseva IO, Lav-
rik OI. Localization of xeroderma pigmentosum group A protein
and replication protein A on damaged DNA in nucleotide exci-
sion repair. Nucleic Acids Res. 2011; 38(22): 8083–94.
91. Krasikova YS, Rechkunova NI, Maltseva EA, Petruseva IO, Silni-
kov VN, Zatsepin TS, Oretskaya TS, Scharer OD, Lavrik OI. In-
teraction of nucleotide excision repair factors XPC-HR23B, XPA,
and RPA with damaged DNA. Biochemistry (Mosc). 2008; 73
(8):886–96.
92. Min JH, Pavletich NP. Recognition of DNA damage by the Rad4
nucleotide excision repair protein. Nature. 2007; 449(7162):
570–5.
93. Krasikova YS, Rechkunova NI, Maltseva EA, Pestryakov PE,
Petruseva IO, Sugasawa K, Chen X, Min JH, Lavrik OI. Compa-
rative analysis of interaction of human and yeast DNA damage
recognition complexes with damaged DNA in nucleotide exci-
sion repair. J Biol Chem. 2013; 288(15):10936–47.
94. Liu Y, Yang Z, Utzat CD, Liu Y, Geacintov NE, Basu AK, Zou Y.
Interactions of human replication protein A with single-stranded
DNA adducts. Biochem J. 2005; 385(Pt 2):519–26.
95. de Laat WL, Appeldoorn E, Sugasawa K, Weterings E, Jaspers
NG, Hoeijmakers JH. DNA-binding polarity of human replica-
tion protein A positions nucleases in nucleotide excision repair.
Genes Dev. 1998; 12(16):2598–609.
96. Maltseva EA, Rechkunova NI, Petruseva IO, Vermeulen W,
Scharer OD, Lavrik OI. Crosslinking of nucleotide excision re-
pair proteins with DNA containing photoreactive damages. Bio-
org Chem. 2008; 36(2):77–84.
97. Bergink S, Toussaint W, Luijsterburg MS, Dinant C, Alekseev S,
Hoeijmakers JH, Dantuma NP, Houtsmuller AB, Vermeulen W.
Recognition of DNA damage by XPC coincides with disruption
of the XPC-RAD23 complex. J Cell Biol. 2012;196(6):681–8.
98. Anin MF, Leng M. Distortions induced in double-stranded oligo-
nucleotides by the binding of cis- or trans-diammine-dichloro
platinum (II) to the d(GTG) sequence. Nucleic Acids Res. 1990;
18(15):4395–400.
99. Bellon SF, Coleman JH, Lippard SJ. DNA unwinding produced
by site-specific intrastrand cross-links of the antitumor drug cis-
diamminedichloroplatinum(II). Biochemistry. 1991; 30(32):
8026–35.
100. van Garderen CJ, van Houte LP. The solution structure of a DNA
duplex containing the cis-Pt(NH3)2[d(-GTG-)-N7(G),N7(G)]
adduct, as determined with high-field NMR and molecular me-
chanics/dynamics. Eur J Biochem. 1994; 225(3):1169–79.
101. Calsou P, Frit P, Salles B. Double strand breaks in DNA inhibit
nucleotide excision repair in vitro. J Biol Chem. 1996; 271(44):
27601–7.
102. Evdokimov AN, Petruseva IO, Pestryakov PE, Lavrik OI. Photo-
activated DNA analogs of substrates of the nucleotide excision
repair system and their interaction with proteins of NER-com-
petent extract of HeLa cells. Synthesis and application of long
model DNA. Biochemistry (Mosc). 2011; 76(1):157–66.
103. Huang JC, Zamble DB, Reardon JT, Lippard SJ, Sancar A.
HMG-domain proteins specifically inhibit the repair of the ma-
jor DNA adduct of the anticancer drug cisplatin by human exci-
sion nuclease. Proc Natl Acad Sci USA. 1994; 91(22):10394–8.
104. Langie SA, Cameron KM, Waldron KJ, Fletcher KP, von Zgli-
nicki T, Mathers JC. Measuring DNA repair incision activity of
mouse tissue extracts towards singlet oxygen-induced DNA da-
mage: a comet-based in vitro repair assay. Mutagenesis. 2011;
26(3):461–71.
105. Araujo SJ, Tirode F, Coin F, Pospiech H, Syvaoja JE, Stucki M,
Hubscher U, Egly JM, Wood RD. Nucleotide excision repair of
DNA with recombinant human proteins: definition of the mi-
nimal set of factors, active forms of TFIIH, and modulation by
CAK. Genes Dev. 2000; 14(3):349–59.
106. Shell SM, Hawkins EK, Tsai MS, Hlaing AS, Rizzo CJ, Chazin
WJ. Xeroderma pigmentosum complementation group C protein
(XPC) serves as a general sensor of damaged DNA. DNA Repair
(Amst). 2013; 12(11):947–53.
107. Vaisman A, McDonald JP, Huston D, Kuban W, Liu L, Van Hou-
ten B, Woodgate R. Removal of misincorporated ribonucleo-
tides from prokaryotic genomes: an unexpected role for nucleoti-
de excision repair. PLoS Genet. 2013; 9(11):e1003878.
Received 15.01.14
183
MODEL DNA FOR INVESTIGATION OF MECHANISM OF NUCLEOTIDE EXCISION REPAIR
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