Synthesis of model DNA and their application as substrates of nucleotide excision repair
Aim. Nucleotide excision repair (NER) is DNA repair system responsible to remove bulky lesions from DNA. These lesions appear in DNA as consequence of UV-light irradiation or environmental stress. Study of NER is extremely important to improve action of chemotherapeutic drugs. Methods. In vitro NER-...
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Інститут молекулярної біології і генетики НАН України
2012
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| Цитувати: | Synthesis of model DNA and their application as substrates of nucleotide excision repair / A.N. Evdokimov, A.Yu. Tsidulko, I.O. Petruseva, L.S. Koroleva, I.Yu. Serpokrylova, V.N. Silnikov, O.I. Lavrik // Вiopolymers and Cell. — 2012. — Т. 28, № 3. — С. 212–217. — Бібліогр.: 20 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1568452025-02-09T17:09:59Z Synthesis of model DNA and their application as substrates of nucleotide excision repair Синтез модельних ДНК та їхнє застосування як субстратів ексцизійної репарації нуклеотидів Синтез модельных ДНК и их применение в качастве субстратов эксцизионной репарации нуклеотидов Evdokimov, A.N. Tsidulko, A.Yu. Petruseva, I.O. Koroleva, L.S. Serpokrylova, I.Yu. Silnikov, V.N. Lavrik, O.I. Structure and Function of Biopolymers Aim. Nucleotide excision repair (NER) is DNA repair system responsible to remove bulky lesions from DNA. These lesions appear in DNA as consequence of UV-light irradiation or environmental stress. Study of NER is extremely important to improve action of chemotherapeutic drugs. Methods. In vitro NER-assay and photoaffinity modification were used. Results. Long linear DNA analogs mimicking NER substrates have been synthesized. DNA analogs are 137-mer duplexes containing in their internal positions nucleotides with bulky substitutes imitating lesions with fluorochloroazidopyridyl and fluorescein groups introduced using spacer fragments at the 4N and 5C positions of dCMP and dUMP (Fap-dC- and Flu-dU-DNA) and DNA containing a (+)-cis-stereoisomer of benzo[a]pyrene-N2-deoxyguanosine (BP-dG-DNA). The interaction of the modified DNA duplexes with the proteins of NER-competent HeLa extract was investigated. The substrate properties of the model DNA in the reaction ofspecific excision were shown to vary in the row Fap-dC-DNA << Flu-dU-DNA < BP-dG-DNA. Conclusions. In vitro assay show that DNA analogs represent an interesting tool for the estimation of cellular repair activities. The developed approach should be of general use forthe incorporation of NER-sensitive distortions into model DNA and seems to be very promising for repair mechanism studies. Keywords: nucleotide excision repair, model bulky substituted DNA substrates. Мета. Ексцизійна репарація нуклеотидів (NER) – це система репарації ДНК, відповідальна за видалення об’ємних пошкоджень зі складу ДНК. Такі пошкодження можуть виникати за впливу як опромінення ультрафіолетом, так і факторів довкілля. Вивчення системи NER є вкрай важливим для підвищення ефективності хіміотерапевтичних препаратів. Методи. Використано реакцію NER in vitro та фотоафінну модификацію. Результати. Синтезовано довгі лінійні ДНК, які імітують субстрати NER, що являють собою 137-мірні ДНК-дуплекси і містять у внутрішніх положеннях ланцюгів нуклеотиди із введеними за допомогою спейсерних фрагментів по 4N- і 5C-положеннях dC і dU фторхлоразидопіридильною і флуоресцеїновою групами (Fap-dC- і Flu-dU- ДНК), а такожДНК, яка вміщує (+)-цис-стереоізомер бензо[a]пі рен-N2- дезоксигуанозину. Досліджено взаємодію модифікованих ДНК-дуплексів з білками NER-компетентного екстракту клітин HeLa. Показано, що субстратні властивості модельних ДНК у реакції специфічної ексцизії змінюються в ряду Fap-dC-ДНК << Flu-dU-ДНК < BP-dG-ДНК. Висновки. Дослідженнями in vitro встановлено, що ДНК-аналоги є важливим інструментом для оцінки клітинної репарації. Розроблений підхід виявився універсальним для включення до складу ДНК пошкоджень, які упізнаються системою NER, а також досить перспективним для вивчення механізмів репарації. Ключові слова: ексцизійна репарація нуклеотидів, модельні ДНК-субстрати. Цель. Эксцизионная репарация нуклеотидов (NER) – это система репарации ДНК, отвечающая за удаление объемных повреждений из состава ДНК. Такие повреждения могут появляться под воздействием как облучения ультрафиолетом, так и факторов окружающей среды. Изучение системы NER крайне важно для повышения эффективности химиотерапевтических препаратов. Методы. Использованы реакция NER in vitro и фотоаффинная модификаця. Результаты. Синтезированы протяженные линейные ДНК, имитирующие субстраты NER, которые представляют собой 137-мерные ДНК-дуплексы, содержащие во внутренних положениях цепей нуклеотиды с введенными с помощью спейсерных фрагментов по 4N- и 5C-положениям dC и dU фторхлоразидопиридильной и флуоресцеиновой группировками (Fap-dC- и Flu-dU- ДНК), а также ДНК, включающая (+)-цис-стереоизомер бензо[a]пирен-N2-дезоксигуанозина. Исследовано взаимодействие модифицированных ДНК-дуплексов с белками NER-компетентно го экстракта клеток HeLa. Показано, что субстратные свойства модельных ДНК в реакции специфической эксцизии меняются в ряду Fap-dC-ДНК << Flu-dU-ДНК < BP-dG-ДНК. Выводы. Исследованиями in vitro установлено, что ДНК- аналоги являются важным инструментом для оценки клеточной репарации. Разработанный подход оказался универсальным для включения в состав ДНК повреждений, узнаваемых системой NER, а также достаточно перспективным для изучения механизмов репарации. Ключевые слова: эксцизионная репарация нуклеотидов, модельные ДНК-субстраты. 2012 Article Synthesis of model DNA and their application as substrates of nucleotide excision repair / A.N. Evdokimov, A.Yu. Tsidulko, I.O. Petruseva, L.S. Koroleva, I.Yu. Serpokrylova, V.N. Silnikov, O.I. Lavrik // Вiopolymers and Cell. — 2012. — Т. 28, № 3. — С. 212–217. — Бібліогр.: 20 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000050 https://nasplib.isofts.kiev.ua/handle/123456789/156845 577.213.3 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Structure and Function of Biopolymers Structure and Function of Biopolymers |
| spellingShingle |
Structure and Function of Biopolymers Structure and Function of Biopolymers Evdokimov, A.N. Tsidulko, A.Yu. Petruseva, I.O. Koroleva, L.S. Serpokrylova, I.Yu. Silnikov, V.N. Lavrik, O.I. Synthesis of model DNA and their application as substrates of nucleotide excision repair Вiopolymers and Cell |
| description |
Aim. Nucleotide excision repair (NER) is DNA repair system responsible to remove bulky lesions from DNA. These lesions appear in DNA as consequence of UV-light irradiation or environmental stress. Study of NER is extremely important to improve action of chemotherapeutic drugs. Methods. In vitro NER-assay and photoaffinity modification were used. Results. Long linear DNA analogs mimicking NER substrates have been synthesized. DNA analogs are 137-mer duplexes containing in their internal positions nucleotides with bulky substitutes imitating lesions with fluorochloroazidopyridyl and fluorescein groups introduced using spacer fragments at the 4N and 5C positions of dCMP and dUMP (Fap-dC- and Flu-dU-DNA) and DNA containing a (+)-cis-stereoisomer of benzo[a]pyrene-N2-deoxyguanosine (BP-dG-DNA). The interaction of the modified DNA duplexes with the proteins of NER-competent HeLa extract was investigated. The substrate properties of the model DNA in the reaction ofspecific excision were shown to vary in the row Fap-dC-DNA << Flu-dU-DNA < BP-dG-DNA. Conclusions. In vitro assay show that DNA analogs represent an interesting tool for the estimation of cellular repair activities. The developed approach should be of general use forthe incorporation of NER-sensitive distortions into model DNA and seems to be very promising for repair mechanism studies.
Keywords: nucleotide excision repair, model bulky substituted DNA substrates. |
| format |
Article |
| author |
Evdokimov, A.N. Tsidulko, A.Yu. Petruseva, I.O. Koroleva, L.S. Serpokrylova, I.Yu. Silnikov, V.N. Lavrik, O.I. |
| author_facet |
Evdokimov, A.N. Tsidulko, A.Yu. Petruseva, I.O. Koroleva, L.S. Serpokrylova, I.Yu. Silnikov, V.N. Lavrik, O.I. |
| author_sort |
Evdokimov, A.N. |
| title |
Synthesis of model DNA and their application as substrates of nucleotide excision repair |
| title_short |
Synthesis of model DNA and their application as substrates of nucleotide excision repair |
| title_full |
Synthesis of model DNA and their application as substrates of nucleotide excision repair |
| title_fullStr |
Synthesis of model DNA and their application as substrates of nucleotide excision repair |
| title_full_unstemmed |
Synthesis of model DNA and their application as substrates of nucleotide excision repair |
| title_sort |
synthesis of model dna and their application as substrates of nucleotide excision repair |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2012 |
| topic_facet |
Structure and Function of Biopolymers |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/156845 |
| citation_txt |
Synthesis of model DNA and their application as substrates of nucleotide excision repair / A.N. Evdokimov, A.Yu. Tsidulko, I.O. Petruseva, L.S. Koroleva, I.Yu. Serpokrylova, V.N. Silnikov, O.I. Lavrik // Вiopolymers and Cell. — 2012. — Т. 28, № 3. — С. 212–217. — Бібліогр.: 20 назв. — англ. |
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Вiopolymers and Cell |
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212
STRUCTURE AND FUNCTION OF BIOPOLYMERS
UDC 577.213.3
Synthesis of model DNA and their application
as substrates of nucleotide excision repair
A. N. Evdokimov1, A. Yu. Tsidulko1, 2, I. O. Petruseva1, L. S. Koroleva1, 2,
I. Yu. Serpokrylova1, V. N. Silnikov1, O. I. Lavrik1, 2
1Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
8, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090
2Novosibirsk State University
2, Pirogova Str., Novosibirsk, Russian Federation, 630090
an_evdokimov@mail.ru
Aim. Nucleotide excision repair (NER) is DNA repair system responsible to remove bulky lesions from DNA.
These lesions appear in DNA as consequence of UV-light irradiation or environmental stress. Study of NER is
extremely important to improve action of chemotherapeutic drugs. Methods. In vitro NER-assay and photoaffi-
nity modification were used. Results. Long linear DNA analogs mimicking NER substrates have been synthe-
sized. DNA analogs are 137-mer duplexes containing in their internal positions nucleotides with bulky substi-
tutes imitating lesions with fluorochloroazidopyridyl and fluorescein groups introduced using spacer fragments
at the 4N and 5C positions of dCMP and dUMP (Fap-dC- and Flu-dU-DNA) and DNA containing a (+)-cis-ste-
reoisomer of benzo[a]pyrene-N2-deoxyguanosine (BP-dG-DNA). The interaction of the modified DNA duplexes
with the proteins of NER-competent HeLa extract was investigated. The substrate properties of the model DNA
in the reaction of specific excision were shown to vary in the row Fap-dC-DNA << Flu-dU-DNA < BP-dG-DNA.
Conclusions. In vitro assay show that DNA analogs represent an interesting tool for the estimation of cellular
repair activities. The developed approach should be of general use for the incorporation of NER-sensitive distor-
tions into model DNA and seems to be very promising for repair mechanism studies.
Keywords: nucleotide excision repair, model bulky substituted DNA substrates.
Intoduction. In eukaryotic cells, the nucleotide exci-
sion repair (NER) is performed through coordinated ac-
tion of multicomponent protein complexes of variable
composition that are sequentially assembled in a dama-
ged DNA region. The main NER enzymes and protein
factors have been identified, but the architecture of spe-
cific protein-nucleic acid complexes, the mechanism of
their functioning, and interaction of their components
are still subjects of active investigation.
NER system recognizes and removes from DNA a
wide variety of structurally diverse helix-distorting bul-
ky adducts. Such lesions appear as a result of exposure
of DNA to various physical or chemical factors (UV or
ionizing radiation, environmental pollutants, chemothe-
rapeutic agents, etc.). During eukaryotic NER a region
with damaged link (24–32 nucleotides) is removed from
DNA followed by reconstruction of the nucleotide se-
quence using an undamaged DNA chain as a template
for the reparative synthesis [1–3].
NER is characterized by broad substrate specifici-
ty. However, the rates of repair of structurally similar
lesions may be substantially different. The efficiency
of removal of the DNA region with a damaged nucleo-
tide estimated using model substrates, in combination
with the results of analysis of interaction between subst-
rate analogs and NER proteins, provides information
on the details of lesion recognition by NER system.
Many biochemical approaches to NER investiga-
tion are based on synthetic substrate analogs, i. e. doub-
le-stranded DNA bearing modification in the defined
position of the molecule, and structural analogs of the
intermediates that appear during NER [4–8]. This fact
determines the interest in development and improve-
ment of methods of model lesions synthesis and crea-
ISSN 0233–7657. Biopolymers and Cell. 2012. Vol. 28. N 3. P. 212–217
Institute of Molecular Biology and Genetics, NAS of Ukraine, 2012
tion of synthetic DNA on their basis. It is known that a
model lesion must be included in long DNA for the
formation of functional NER complexes [9]. In some
works, substrate properties were studied using circular
structures based on a phage or plasmid DNA and bea-
ring lesions in the target position (or positions) or sta-
tistically introduced into the DNA molecule at a parti-
cular frequency. This approach demands sufficient ef-
forts to be applied.
Another type of model structures being substrates
of the eukaryotic NER system is linear DNA duplexes
of > 120 bp in length bearing a modified nucleotide in
an internal position of the chain at approximately equal
distances from the ends of the duplex [10]. The use of
long DNA analogs of substrates containing regions of
several unpaired bases, including those introduced at a
distance from the lesion, made it possible to specify the
role of the undamaged chain in lesion recognition and
to confirm the hypothesis of a two-stage (or bipartite)
[11, 12] mechanism of lesion recognition. For instance,
it has been shown that the efficiency of repair of model
DNA containing hard-to-repair cyclobutane pyrimidi-
ne dimers noticeably increases on introduction of a bub-
ble or a loop of three unpaired bases in size on the 5'-si-
de of the lesion [12]. Besides, it was recently shown by
the example of long linear DNA containing a benz[a]py-
rene modification that lesion recognition by NER pro-
teins and efficiency of lesion removal depend not only
on the type of modification introduced but also on de-
tails such as differences in the spatial structure of the le-
sion [13] and sequence of the regions flanking the le-
sion [14].
The goal of the present work was to create long linear
DNA analogs of NER substrates and then to analyze
their interaction with proteins of cell extracts containing
a complex of NER proteins and to estimate the proper-
ties of the created model DNA as substrates of the NER.
Materials and methods. Fluorescein-5(6)-carbo-
xamidocaproyl-[5-(3-aminoallyl)-2'-deoxyuridine-5'-
triphosphate] (Flu-dUTP) was from Biosan (Novosi-
birsk). Exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloro-
pyridine-6-yl)-3-aminopropionyl]aminoethyl}-2'-de-
oxycytidine-5'-triphosphate (Fap-dCTP) was synthe-
sized as described in [15] and kindly provided by S. V.
Dezhurov. Deoxyribooligonucleotide containing (+)-
cis-benzo[a]pyrene-N2-deoxyguanosine (BP-dG) has
been kindly provided by N. E. Geatsintov (New York
University, USA).
Deoxyribooligonucleotides (ONT) and [α-32P]dCTP
(3000 Ci/mmol) produced at the Institute of Chemical
Biology and Fundamental Medicine, Siberian Branch of
the Russian Academy of Sciences.
Proteins preparation. NER-competent extract of
HeLa cells was obtained by the method described in
[10]. Recombinant heterodimer XPC-HR23B (Flag-
XPC and in 6His-Tag-HR23B) was obtained by the me-
thod described in [16] with slight modifications.
Synthesis of model DNA was described in details
in [17]. Base substituted dNTP analogs (Fap-dCTP and
Flu-dUTP) were used as substrates of DNA polymerase
to introduce modified dNMP moiety into the 3'-end of
ONT. Long modified ONT was synthesized using liga-
tion of modified 5'-component with radioactively labe-
led 3'-component as described earlier [17].
For the formation of model DNA duplexes modifi-
ed long ONT and complementary unmodified ONT we-
re annealed.
The BP-modified DNA chain was obtained by liga-
tion of 11-nt 5-[32P]-ONT, bearing BP-dG and flanking
ONTs.
In vitro NER assay. The efficiency of removal of
DNA region containing model lesions was described in
details in [17] and determined using both direct and in-
direct («fill-in») methods. For direct detection reaction
mixtures containing of internally labeled model DNA
duplexes were incubated with NER-competent cell ex-
tract. 1 × NER buffer (25 mM Hepes, pH 7.8, 45 mM
NaCl, 4.4 mM MgCl2, 0.1 mM EDTA, 4 mM ATP. Re-
action mixtures were analyzed by electrophoresis in po-
lyacrylamide gel under denaturing conditions followed
by quantitative autoradiography. Quantitative analysis
was done relatively to total radioactivity in each lane,
using control lane signal as baseline.
For «fill-in» synthesis reaction mixtures was addi-
tionally contain the template 5'-gggggctcggcaccgtcacc
ctggatgctgtagg-p-3', and the model DNA duplex was
unlabeled. After incubation with NER-competent cell
extract, radioactive label was introduced into excised
DNA fragments using [α-32P]dCTP as substrate of
DNA polymerase reaction.
The effect of Fap-dC- and Flu-dU-DNA on the le-
vel of specific excision from [32P]-BP-dG-DNA was stu-
213
SYNTHESIS OF MODEL DNA AND THEIR APPLICATION AS SUBSTRATES
died. The reaction mixtures containing [32P]-BP-dG-
DNA were incubated with NER-competent cell extract
in the absence or in the presence of equimolar unlabe-
led competitive Fap-dC- or Flu-dU-DNA. The analysis
of samples was carried out as described above.
Results and discussion. Using the improved enzy-
matic method of synthesis, we have synthesized long
Fap-dC- and Flu-dU-DNA (137 bp) and analyzed their
properties as substrates for the excision reaction cata-
lyzed by the proteins of NER-competent extract of
HeLa cells. BP-dG-DNA (131 bp) synthesized with the
respective modified ONT was used as a standard subst-
rate. The structures of model DNAs, containing bulky
lesions and schematic representation of the DNA frag-
ments containing modifications are shown in Fig. 1.
The activity of these DNA analogs as NER substra-
tes were demonstrated at the stage of lesion removal.
The activity was measured by two methods: direct and
indirect detection of the excision products (see Mate-
rials and methods). During the direct detection, Fap-
dC-DNA and Flu-dU-DNA contained a 32P-label imme-
diately in the damaged nucleotide (on the 3'-side); in
the case of BP-DNA, the label was located at a distance
of five nucleotides on the 5'-side of the lesion. Thus,
excision products (22–32-nucleotide DNA fragments
bearing lesions) must contain 32P-label. The advantage
of this method is the possibility to quantitatively assess
lesion removal efficiency without additional stages.
DNA modifications induced by benzo[a]pyrene are
one of the most widespread and well-studied types of
lesions repaired by NER in humans [1–3, 14]. This mo-
del lesion belongs to a class of systematically studied
and rather effective synthetic NER substrates. How-
ever, it should be noted that the level of excision in ex-
periments in vitro rarely exceeds 5 % even for such mo-
del DNA [13]. During the incubation of [32P]-BP-DNA
with the proteins of NER-competent HeLa extract, ra-
dioactive DNA fragments of 22–32 nucleotides were
observed in the mixture. The quantity of specific hydro-
lysis products increased during 10–60 min from the be-
ginning of the reaction. Excised fragments accumula-
tion is also dependent on the concentration of extract
proteins in the reaction mixture within the analyzed
range of concentrations (2–15 µg/µl) (data not shown).
Fig. 2, a, presents the results of the analysis of reaction
mixture after 40 min of incubation of [32P]-BP-DNA
with the extract proteins. The level of excision of da-
maged fragments from [32P]-Fap-dC- and [32P]-Flu-dU-
DNA was not sufficient to detect the excision reaction
by using this method.
Appearance of fragments of typical length from
Flu-dU-DNA was detected using the «fill-in» method
214
EVDOKIMOV A. N., TSIDULKO A., PETRUSEVA I. O. ET AL.
P
O
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O
O
O
OH
O P
N
OH
OH
OH
O
O
NH
NH
N
N
O
N
N
O
NH
OH
N
H
O
N
H
N
F
Cl
F O ppp
N3
O
N
NH
O
OH
O
O
N
H
O
N
H
O
O OHOH
O
O
ppp
a
b
c
Fig. 1. Model lesions. Structures of Fap-dCTP (a) and Flu-dUTP (b) bearing bulky lesion and the fragment of deoxyribooligonucleotide contai-
ning (+)-cis-benzo[a]pyrene-N2-deoxyguanosine (c) used for the synthesis of modified DNA chains. BP-modified oligonucleotide has been kind-
ly provided by N. E. Geatsintov (New York University, USA)
(Fig. 2, b). Radioactive [α-32P]dCMP was introduced
as a label into the removed DNA fragments with da-
maged nucleotides using DNA-polymerase reaction
and complementary template ONT. The advantage of
such estimation of substrate properties is higher sensi-
tivity and absence of necessity of synthesis of model
DNA with high specific radioactivity. However, pro-
ducts of specific excision from Fap-dC-DNA were not
revealed by this method either.
Flu-dU- and Fap-dC-DNA demonstrated the ability
for effective competition with BP-DNA for the interac-
tion with proteins of the cell extract excision complex.
The experiments (results are shown in Fig. 3) were per-
formed using [32P]-BP-DNA and nonradioactive com-
petitive DNA duplexes. In the presence of an equimolar
amount of the control unmodified duplex, the level of
excision for [32P]-BP-DNA was no less than 95 % of the
level of excision observed in the absence of competiti-
ve DNA. As a result of adding the equimolar amount of
Flu-dU- or Fap-dC-DNA to the reaction mixture, the le-
vel of excision of damaged fragments from [32P]-BP-
DNA significantly decreased. In the presence of Fap-
dC-DNA, the level of excision from BP-DNA was
lower by 20 %, while the presence of Flu-dU-DNA
reduced the level of BP-DNA specific hydrolysis two-
fold. Decrease in the quantity of radioactive cleavage
products is evidently due to distribution of NER pro-
teins between radioactive BP-DNA and nonradioactive
Fap-dC- or Flu-dU-DNA.
Our results, in combination with the data of pre-
vious studies [18, 19], allow us to consider the linear
137-bp Fap-dC-DNA as unrepairable NER substrates.
Unrepairable substrate analogs, i. e. structures capable
of selective binding of DNA repair proteins but not
liable to further processing, are most dangerous for
cells. Accumulation of such DNA can considerably re-
duce the efficiency of the repair machinery due to im-
mobilization of its protein components within nonpro-
ductive nucleoprotein complexes [20]. The ability of
model lesion to effectively inhibit the repair process in
vitro indicates the possibility of its application as a pro-
totype for creation of selective DNA repair inhibitors
contributing to the enhancement of efficiency in che-
motherapy.
In additional this DNA analog can be applied as ef-
fective photoaffinity probe to analyze interaction of
NER proteins with bulky DNA lesion. The data speak in
favor possibility to use this photoactive DNA to cross-
link XPC-HR23B (Fig. 4).
Our preliminary studies with novel non-nucleoside
adducts which contain N-[6-(9-anthracenylcarbamoyl)
hexanoyl]-3-amino-1,2-propandiol and N-[6-(5(6)- fluo-
resceinylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol
residues indicated their extremely high substrate pro-
215
SYNTHESIS OF MODEL DNA AND THEIR APPLICATION AS SUBSTRATES
1 2 M
← 34
← 30
← 22
← 19
nt
M
BP-DNA
34 30 22 nt
← 30
← 22
nt
1 2 M
M
30 22 nt
Flu-dU-DNA
ba
Fig. 2. Radioautographs of gels after separation of excision reaction products: a – direct detection of excision products (1 – analysis of reaction
mixture containing 20 nM [32P]-BP-DNA and 7 µg/µl of the HeLa extract proteins in 1 × NER buffer incubated for 40 min at 30 °C; 2 – modified
duplex incubated without the extract); b – indirect detection of excision products; reaction mixtures contained nonradioactive 20 nM Fap-dC-DNA
or Flu-dU-DNA and 7 µg/µl of the HeLa extract proteins; excision products fused with the respective template were completed using Taq polymerase
with [α-32P]dCTP as a substrate (1 – unmodified DNA; 2 – Fap-dC-DNA; 3 – Flu-dU-DNA); M – markers of DNA. On the right, a and b panels show
the results of densitometric analysis of the radioautographs
EVDOKIMOV A. N., TSIDULKO A., PETRUSEVA I. O. ET AL.
perties in NER reaction in vitro. Such model lesion seems
to be very promising to study DNA repair mechanism.
To conclude, it should be noted, that developed ap-
proach is of general use for the incorporation of NER-
sensitive distortions into model DNA. Many types of
NER substrates can be created by this way, including
DNA with photoactivated and fluorescent groups. The
new artificial DNA containing bulky non-nucleoside
modifications will be useful for fundamental and me-
dical research as a tool to detect activity of NER sys-
tem in cells.
Acknowledgements. This work was supported by the
Russian Foundation for Basic Research (project N 12-
04-00487) and by RAS Program «Molecular and Cel-
lular Biology».
О. М. Євдо ки мов, О. Ю. Ци дул ко, І. О. Пет ру се ва, Л. С. Ко роль о ва,
І. Ю. Сер пок ри ло ва, В. М. Силь ни ков, О. І. Лав рик
Син тез мо дель них ДНК та їхнє за сто су ван ня як суб стратів
ек сцизійної ре па рації нук ле о тидів
Ре зю ме
Мета. Ексцизійна ре па рація нук ле о тидів (NER) – це сис те ма ре -
па рації ДНК, відповідаль на за ви да лен ня об’ємних по шкод жень зі
скла ду ДНК. Такі по шкод жен ня мо жуть ви ни ка ти за впли ву як
опроміне ння ультрафіоле том, так і фак торів довкілля. Вив чен ня
сис те ми NER є вкрай важ ли вим для підви щен ня ефек тив ності
хіміот е ра пев тич них пре па ратів. Ме то ди. Ви ко рис та но ре акцію
NER in vitro та фо то афінну мо ди фи кацію. Ре зуль та ти. Син те зо-
вано довгі лінійні ДНК, які іміту ють суб стра ти NER, що яв ля ють
со бою 137-мірні ДНК-дуп лек си і містять у внутрішніх по ло жен-
нях лан цюгів нук ле о ти ди із вве де ни ми за до по мо гою спей сер них
фраг ментів по 4N- і 5C-по ло жен нях dC і dU фтор хло ра зи допіри -
диль ною і флу о рес цеї но вою гру па ми (Fap-dC- і Flu-dU- ДНК), а
та кож ДНК, яка вміщує (+)-цис-сте реоізо мер бен зо[a]пі рен-N2-
дез окси гу а но зи ну. Дослідже но взаємодію мо дифіко ва них ДНК-
дуплексів з білка ми NER-ком пе тен тно го екстрак ту клітин HeLa.
По ка за но, що суб стратні влас ти вості мо дель них ДНК у ре акції
специфічної ек сцизії зміню ють ся в ряду Fap-dC-ДНК << Flu-dU-
ДНК < BP-dG-ДНК. Вис нов ки. Досліджен ня ми in vitro вста нов -
ле но, що ДНК-ана ло ги є важ ли вим інстру мен том для оцінки клі-
тин ної ре па рації. Роз роб ле ний підхід ви я вив ся універ саль ним для
вклю чен ня до скла ду ДНК по шкод жень, які упізна ють ся сис те -
мою NER, а та кож до сить пер спек тив ним для вив чен ня ме ханіз-
мів ре па рації.
Клю чові сло ва: ек сцизійна ре па рація нук ле о тидів, мо дельні
ДНК-суб стра ти.
216
a b
131→
1 2 M 3 4 5
34→
30→
22→
M, nt
34
30
34
M
2
3 4
5
Fig. 3. Radioautograph (a) of gel after separa-
tion of excision reaction products by electro-
phoresis under denaturing conditions (10 %
polyacrylamide gel, 7 M urea). Reaction mix-
tures containing 20 nM [32P]-BP-DNA and
1 × NER buffer were incubated for 40 min at
30 °C in the absence (2) or presence of 20 nM
unlabeled competitive DNA (3 – unmodifi-
ed duplex; 4 – Flu-dU-DNA; 5 – Fap-dC-
DNA) and 7 µg/µl of the HeLa extract pro-
teins; 1 – results of separation of the mixture
containing only unmodified duplex stored with
the extract proteins; M – markers of DNA
length (nt); b – the result of densitometric
analysis
1 2
– +
250 →
150 →
100 →
75 →
Benzonase
Fig. 4. Radioautograph of gel after separation of proteins to DNA pho-
tolinking products. Reaction mixtures contained 10 nM [32P]-Fap-dC-
DNA and 0.5 M XPC-HR23B. The mixtures were exposed to UV ra-
diation at 312 nm and intensity of 3 J/cm2⋅min for 5 min. After the UV
exposure, aliquots taken from each mixture were treated with benzo-
nase endonuclease (1 activity unit per 10 µl of a sample, 30 min at 37 °C).
The reaction was stopped by heating with the denaturing buffer for
protein samples. The buffer for samples was also added to the residual
part of the reaction mixture: 1 – sample not treated with benzonase; 2 –
sample treated with benzonase. Products of photolinking were separated
by electrophoresis in 10 % polyacrylamide gel according to Laemmli
А. Н. Евдо ки мов, А. Ю. Ци дул ко, И. О. Пет ру се ва, Л. С. Ко ро ле ва,
И. Ю. Сер пок ры ло ва, В. Н. Силь ни ков, О. И. Лав рик
Син тез мо дель ных ДНК и их при ме не ние в ка час тве суб стра тов
экс ци зи он ной ре па ра ции нук ле о ти дов
Ре зю ме
Цель. Эксци зи он ная ре па ра ция нук ле о ти дов (NER) – это сис те ма
ре па ра ции ДНК, от ве ча ю щая за уда ле ние об ъ ем ных по вреж де -
ний из со ста ва ДНК. Та кие по вреж де ния мо гут по яв лять ся под
воз де йстви ем как об лу че ния ультра фи о ле том, так и фак то ров
окру жа ю щей сре ды. Изу че ние сис те мы NER край не важ но для
по вы ше ния эф фек тив нос ти хи ми о те ра пев ти чес ких пре па ра -
тов. Ме то ды. Исполь зо ва ны ре ак ция NER in vitro и фо то аф фин-
ная мо ди фи ка ця. Ре зуль та ты. Син те зи ро ва ны про тя жен ные ли-
не й ные ДНК, ими ти ру ю щие суб стра ты NER, ко то рые пред став -
ля ют со бой 137-мер ные ДНК-дуп лек сы, со дер жа щие во внут рен -
них по ло же ни ях це пей нук ле о ти ды с вве ден ны ми с по мощью спей-
сер ных фраг мен тов по 4N- и 5C-по ло же ни ям dC и dU фтор хлор-
ази до пи ри диль ной и флу о рес це и но вой груп пи ров ка ми (Fap-dC- и
Flu-dU- ДНК), а так же ДНК, вклю ча ю щая (+)-цис-сте ре о и зо мер
бензо[a]пи рен-N2-дез окси гу а но зи на. Иссле до ва но вза и мо де йствие
мо ди фи ци ро ван ных ДНК-дуп лек сов с бел ка ми NER-ком пе тент-
но го экс трак та кле ток HeLa. По ка за но, что субстрат ные свой-
ства мо дель ных ДНК в ре ак ции спе ци фи чес кой экс ци зии ме ня ют -
ся в ряду Fap-dC-ДНК << Flu-dU-ДНК < BP-dG-ДНК. Вы во ды.
Иссле до ва ни я ми in vitro уста нов ле но, что ДНК- ана ло ги яв ля ют ся
важ ным инстру мен том для оцен ки кле точ ной ре па ра ции. Раз ра -
бо тан ный под ход ока зал ся уни вер саль ным для вклю че ния в со -
став ДНК по вреж де ний, узна ва е мых сис те мой NER, а так же до-
ста точ но пер спек тив ным для из уче ния ме ха низмов ре па ра ции.
Клю че вые сло ва: экс ци зи он ная ре па ра ция нук ле о ти дов, мо -
дель ные ДНК-суб стра ты.
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217
SYNTHESIS OF MODEL DNA AND THEIR APPLICATION AS SUBSTRATES
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