Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates
Цель. Исследовать TLS-активность ДНК-полимераз β и λ человека на ДНК-дуплексах, содержащих 5-формилуридин (5-foU) и имитирующих интермедиаты репликации лидирующей цепи геномной ДНК, а также влияние на нее репликативных факторов hRPA и hPCNA. Методы. «Задержка в геле» (EMSA), ферментативная кинетика....
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| Date: | 2012 |
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Інститут молекулярної біології і генетики НАН України
2012
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| Cite this: | Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates / E.A. Belousova, O.I. Lavrik // Вiopolymers and Cell. — 2012. — Т. 28, № 3. — С. 218–222. — Бібліогр.: 21 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860001331349028864 |
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| author | Belousova, E.A. Lavrik, O.I. |
| author_facet | Belousova, E.A. Lavrik, O.I. |
| citation_txt | Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates / E.A. Belousova, O.I. Lavrik // Вiopolymers and Cell. — 2012. — Т. 28, № 3. — С. 218–222. — Бібліогр.: 21 назв. — англ. |
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| description | Цель. Исследовать TLS-активность ДНК-полимераз β и λ человека на ДНК-дуплексах, содержащих 5-формилуридин (5-foU) и имитирующих интермедиаты репликации лидирующей цепи геномной ДНК, а также влияние на нее репликативных факторов hRPA и hPCNA. Методы. «Задержка в геле» (EMSA), ферментативная кинетика. Результаты. Изучена способность ДНК-полимераз β и λ катализировать синтез ДНК через 5-foU. Определены кинетические характеристики этого процесса в отсутствие и в присутствии белковых факторов hRPA и hPCNA. Выводы. Показано, что: 1) оба фермента способны вести TLS на использованных ДНК-субстратах независимо от условий реакции, однако ДНК-полимераза λ оказалась более точным ферментом; 2) hRPA стимулирует эффективность немутагенного TLS, катализируемого ДНК-полимеразой λ, непосредственно при встраивании нуклеотида напротив 5-foU и не влияет на эффективность этого процесса в том случае, когда повреждение сдвинуто в дуплексную часть ДНК; 3) hPCNA не влияет на эффективность TLS, катализируемого обоими ферментами.
Kлючевые слова: синтез ДНК через повреждение, ДНК-полимеразы β и λ, 5-формилуридин.
Мета. Дослідити активність TLS ДНК-полімераз β і λ людини на ДНК-дуплексах, які містять 5-формілуридин (5-foU) та імітують інтермедіати реплікації лідируючого ланцюга геномної ДНК, а також вплив на неї реплікативних факторів hRPA и hPCNA. Методи. «Затримка в гелі» (EMSA), ферментативна кінетика. Результати. Вивчено здатність ДНК-полімераз β і λ активувати синтез ДНК через 5-foU. Визначено кінетичні характеристики цього процесу за відсутності та в присутності білкових факторів hRPA і hPCNA. Висновки. Показано, що 1) обидва ферменти можуть вести TLS на використаних ДНК-субстратах незалежно від умов реакції, однак ДНК-полімераза λ виявилася точнішим ферментом; 2) hRPA стимулює ефективність немутагенного TLS, каталізованого ДНК-полімеразою λ, безпосередньо при вбудовуванні нуклеотиду навпроти 5-foU і не діє на ефективність цього процесу у тому разі, коли пошкодження зсунуте в дуплексну частину ДНК; 3) hPCNA не впливає на ефективність TLS, каталізованого обома ферментами
Ключові слова: синтез ДНК через пошкодження, ДНК-полімерази β і λ, 5-формілуридин.
Aims. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ) using 5-formyluridine (5-foU) containing DNA duplexes which are imitating the intermediates during replication of the leading DNA strand, and to study the influence ofreplication factors hRPA and hPCNA on this activity. Methods. The EMSA and the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis across 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i) both proteins are able to catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate enzyme; (ii) hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol λ at the nucleotide incorporation directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage displaced into the duplex; (iii) hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.
Keywords: translesion synthesis, DNA polymerases β and λ, 5-formyluridine.
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218
UDC 577.151.45; 57.037; 577.325.5
Study of the activity of DNA polymerases β and λ using
5-formyluridine containing DNA substrates
E. A. Belousova, O. I. Lavrik
Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
8, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090
sheffield@ngs.ru
Aims. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ) using 5-formyluridine
(5-foU) containing DNA duplexes which are imitating the intermediates during replication of the leading DNA
strand, and to study the influence of replication factors hRPA and hPCNA on this activity. Methods. The EMSA and
the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis ac-
ross 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of
protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i) both proteins are able to
catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate en-
zyme; (ii) hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol λ at the nucleotide incor-
poration directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage
displaced into the duplex; (iii) hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.
Keywords: translesion synthesis, DNA polymerases β and λ , 5-formyluridine.
Introduction. Nucleobases are chemically modified un-
der the influence of environmental or organic factors.
One of the major product of the thymine oxidation un-
der UVA and ionized irradiation is 5-formyluridine, 5-
foU. The presence of 5-foU in the DNA doesn’t lead to
replication block of both DNA strands, however replica-
tive DNA polymerases such as Klenow fragment, DNA
polymerase alfa and gamma are able to incorporate any
of all 4 dNTPs opposite 5-foU allowing for transition
and transversions [1].
Translesion synthesis is one of the main strategies
which is used by cell for replication of damaged DNA.
It is known, that TLS is catalyzed by special DNA poly-
merases with majority them belonging to the Y-family
[2]. However, some data exists that indicate the role of
X-family DNA polymerases β and λ in TLS [3].
The set of in vitro experiments fulfilled using diffe-
rent damaged DNA substrates indicate that the efficacy
of the nucleotide incorporation opposite the lesion and
the capability to catalyze DNA synthesis passing the le-
sion are varied for different DNA polymerases. Since
TLS occurred directly in the replication complex, such
replicative factors as PCNA and RPA can influence ef-
ficacy and accuracy of the TLS polymerases. Some ex-
periments were already done but the result doesn’t
complete the full relations – in common, PCNA and RPA
influence dependent from type of DNA lesion and spe-
cific DNA polymerase [4].
Therefore, here we attended to investigate the TLS
activity of human DNA polymerases β and λ (pols β and
λ) using DNA duplexes with 5-foU in a template by ki-
netic approaches. As for DNA substrates we used pri-
med DNA duplexes which imitate the TLS interme-
diates during the replication of the leading strand. Reac-
tions were carried out in «high» and «low» levels fi-
delity of polymerization conditions that is in the presen-
ce of Mg2+ or Mn2+ ions, respectively.
Materials and methods. The following substances
were used: BSA («New England Biolabs», USA); phage
T4 polynucleotide kinase (5000 U/ml, «Biosan», Rus-
ISSN 0233–7657. Biopolymers and Cell. 2012. Vol. 28. N 3. P. 218–222
Institute of Molecular Biology and Genetics, NAS of Ukraine, 2012
219
BELOUSOVA E. A., LAVRIK O. I.
sia); [γ-32P]ATP with specific activity 5000 Ci/mmol
(LBT ICBFM SB RAS); dNTP, reagents for electropho-
resis and the main buffers components («Sigma», USA).
Human recombinant pols β and λ were purified from Es-
cherichia coli BL21(DE3) RP cells as described in [5]
and [6], correspondingly. Purified hRPA and hPCNA
were kindly provided by I. O. Petruseva (LBCE ICBFM
SB RAS). The following oligonucleotides were used:
primer strand 5'-(d)CGGTATCCACCAGGTCTG-3'
(«GenSet», Switzerland); template strand 5'-(d)GGCT
TCATCGTTGTCU5-foCAGACCTGGTGGATACCG-
3' (synthesized and kindly provided by T. S. Zatsepin, A.
N. Belozersky Institute of Physico-Chemical Biology,
Moscow State University).
Incorporation of the radioactive label into the 5'-
end of the primer was performed as described in [7].
Determination of the Kd values for the DNA-pro-
tein complexes by EMSA was performed as described
in [8].
dNMP were incorporated into the 3'-end of primer
by DNA pols β and λ. Reactions mixtures (10 µl) con-
tained 0.01 µM 5'-[32P]labeled DNA substrate, 0.01 µM
pol, 5 µM dNTP, 1 mM MgCl2 or MnCl2 in TDB buffer
(50 mM Тris-HCl, рН 8.0, 0.5 mM DTT, 0.25 mg/ml
BSA). All reactions were incubated at 37 oC for 15 min.
The reactions were terminated by placement on ice. Re-
action products were resolved by electrophoresis in
20 % denaturant PAAG as described in [9] and subjected
to autoradiography using Molecular Imager FX («Bio-
Rad», USA). Analysis was performed with the Quan-
tity One («BioRad»).
The Michaelis constants and the maximal rates of
DNA synthesis were determined as described in [10].
The data were fitted according to Michaelis-Menten ki-
netic equation.
The effect of hRPA and hPCNA on the polymeri-
zation reaction catalyzed by pols β and λ was studied
as follow. The reaction mixtures (10 µl) contained 0.01
µM 5'-[32P]labeled DNA substrate, 0.01 µM pol, 0.5–
200 µM dNTP, 1 mM MgCl2 or MnCl2, hRPA or/and
hPCNA in TDB buffer. The hRPA concentration was
5, 10 or 20 nM (in a ratio DNA:hRPA = 2:1, 1:1, 1:2).
The hPCNA concentration was 50, 100 or 200 nM (in a
ratio DNA:hPCNA = 1:5, 1:10, 1:20). The combined ef-
fect of hPCNA and hRPA was investigated by setting
the hPCNA concentration at 100 nM (in a ratio DNA :
: hRPA = 2:1, 1:1, 1:2). The reaction was performed at
37 °C for 15 min and terminated by placement on ice.
The reaction products were analyzed as described above.
Results and discussion. In the present work we stu-
died the TLS activity of pols β and λ on DNA substra-
tes mimicking the TLS intermediates during the replica-
tion of the leading strand. 5-foU located in the +1 po-
sition of the template strand with respect to the 3'-end of
the primer was used as a lesion.
To estimate the affinity of the selected DNA pols to
DNA probes we carried out gel mobility shift assays.
Data obtained indicate that introduction of the 5-foU de-
crease the affinity of pol β to DNA substrate containing
single-stranded protruding 5'-end (Kd ~ 0.18 µM for un-
damaged DNA and ~ 0.53 µM for 5-foU-DNA). The sa-
me modification of the DNA substrate result in decrea-
se of Kd value, i. e. to increase of the pol λ affinity to the
DNA used (0.36 µM for undamaged DNA and ~0.18 µM
for 5-foU-DNA).
To determine which dNMP could be incorporated
in the model system DNA synthesis was carried out in
the presence of either one or all four dNTPs (Figure, B).
It’s easy to see that pol λ was more accurate during DNA
synthesis across 5-foU – the enzyme was able to cataly-
ze the incorporation of dAMP which is the complement
to the template dTMP. Insignificant incorporation of
dGMP in the presence of Mn2+ ions (not more than 5 %)
can be explained by «template slippage» mechanism
clarified for pol λ [11]. In any case, kinetic characte-
ristics of the incorporation of correct nucleotide charac-
terized dATP as more effective substrate (for example,
Km (dATP/Mn2+) ~0.07 µM, Кm (dGTP/Mn2+) ~0.15 µM).
Pol β proved itself as extremely inaccurate enzyme
which is able to catalyze the incorporation of all dNTP
opposite 5-foU (Figure, A). The incorporation of dGMP
can be explained by «template slippage» mechanism
[11], and the incorporation of dAMP and dTMP pro-
bably is the result of a low fidelity of pol β [12]. In any
case, the efficacy of the incorporation of correct dAMP
was much higher with comparison of the incorporation
efficacy of incorrect dGMP (kcat/Km (dATP/Mg2+) ~0.051
µM–1s–1, kcat/Km (dGTP/Mg2+) ~0.005 µM–1s–1).
Thus, independently of the reaction conditions both
enzymes are able to catalyze TLS process using descri-
bed DNA. However, pol β doesn’t have the specificity
at the passage of this type of damage. At the same time,
220
SYNTHESIS BY DNA POLYMERASES β AND λ ACROSS 5-foU
pol λ is able to catalyze the incorporation of correct
dAMP which is the complement to the original dTMP,
thereby maintaining initial DNA sequence and doesn’t
force point mutations.
Since TLS constitutively associated with the repli-
cation machinery, replication and repair proteins could
play a key role during this process. One of the potential
TLS-participant during the replication on the leading
strand is hRPA which contain subunits p70, p32 and
p14 [13]. The main function of RPA include stabiliza-
tion of DNA in a single-stranded conformation, posi-
tioning of the proteins at the nucleic-protein comple-
xes, and activity stimulation of other protein factors.
Here the efficacy of DNA synthesis in the presence
of hRPA was estimated by comparing the kinetic para-
meters for incorporation of correct dAMP or incorrect
dGMP (Tables 1 and 2). As can be seen from presented
results, the presence of hRPA doesn’t influence the TLS
efficacy catalyzed by pol β. Contrariwise, the efficacy
of nonmutagenic TLS catalyzed by pol λ was enhanced
by hRPA by not affecting the dAMP incorporation and
negatively affecting the dGMP incorporation (Table 2).
Interestingly, the presence of hRPA doesn’t influence
on the activity of pol λ during the next step of DNA syn-
thesis, i. e. in the case when 5-foU is located in the 0 po-
sition of the template strand with respect to the 3'-end
of the primer (Table 2).
The next inherent component of the replication ma-
chinery is PCNA. First of all, PCNA is a processivity
factor for the replicative DNA pols. Additionally, it par-
ticipates in the coordination of protein-protein interac-
tion in the replication complex [14]. Moreover, due to
the majority of different proteins interacting with PCNA
it can be suggested as a link between the replication and
other cell’s process such as TLS. Particularly, hPCNA
has been proposed to coordinate switching from replica-
tive DNA pols to specialized TLS enzymes [15]. Fur-
ther, it has been demonstrated that PCNA physically in-
teracts with pols β and λ [16, 17].
The influence of hPCNA on TLS-activity of pols β
and λ was estimated as described above. It turned out,
that hPCNA doesn’t influence the TLS efficacy of both
pols using 5-foU-containing DNA substrates (Tables 1
and 2). These data are consistent with already publi-
shed results: it was found, that PCNA increase the affi-
nity of pol λ to the 3'-end of the primer, stabilize the for-
mation complex between the DNA substrate and enzy-
me, which allows to increase the processivity of DNA
synthesis using undamaged DNA but doesn’t change
the enzyme affinity to dNTP and the efficacy of correct
nucleotide incorporation [18]. At the same time, no alte-
ration of the interplay between pol β and damaged DNA
in the presence of PCNA was obtained [4].
Absence of cumulative effect of hRPA and hPCNA
on TLS-activity of pol β across 5-foU was shown (Tab-
le 1). In contrast, combine presence of two protein fac-
tors lead to increase of accuracy of TLS catalyzed by
DNA pol λ mainly due to the increasing of the incorpo-
ration of correct dAMP (Table 2).
Therefore, hRPA has stimulated the efficacy of non-
mutagenic TLS catalyzed by pol λ at the incorporation
of dNMP opposite to 5-foU and didn’t have influence
C 1 2 3 4 5 6 7 8 9 10
A G T C N A G T C N A G T C N A G T C N
18 nt →
19 nt →
34 nt →
A B
C 1 2 3 4 5 6 7 8 9 10
Mg2+ Mg2+Mn2+ Mn2+
DNA synthesis catalyzed by pols β
(A) and λ (B) using 5-foU-contai-
ning DNA substrates
on the reaction if the synthesis starts from the 0 position
relative to the damage.
Combined effect of hRPA and hPCNA analogously
increase the accuracy of TLS across 5-foU catalyzed by
pol λ mainly due to the increasing of the incorporation
of correct dNMP.
Basically, obtained results are in accordance with
the published data. Indeed, it was shown using 5'-ex-
tended DNA substrates that TLS-activity of pol λ ac-
ross oxidative damages can be modulated by hRPA [4,
19–21]. At the same time, hRPA didn’t display any ef-
fect on the TLS-capacity of pol β across oxidative da-
mages [4].
Combining the obtained results with published data
it can be conclude, that pol λ plays an important role in
TLS across 5-foU during genomic replication on the
leading strand.
Acknowledgements. This work was supported by a
grant from the Russian Foundation for Basic Research
(N12-04-00178-а).
Є. А. Бе ло у со ва, О. І. Лав рик
Активність ДНК-поліме раз β і λ при син тезі ДНК-суб стратів, які
містять 5-формілу ри дин
Ре зю ме
Мета. Дослідити ак тивність TLS ДНК-поліме раз β і λ лю ди ни на
ДНК-дуп лек сах, які містять 5-формілу ри дин (5-foU) та іміту ють
інтер медіати реплікації ліди ру ю чо го лан цю га ге ном ної ДНК, а та-
кож вплив на неї репліка тив них фак торів hRPA и hPCNA. Мето-
ди. «Затрим ка в гелі» (EMSA), фер мен та тив на кіне ти ка. Ре зуль -
та ти. Вив че но здатність ДНК-поліме раз β і λ ак ти ву ва ти син тез
ДНК че рез 5-foU. Виз на че но кіне тичні ха рак те рис ти ки цьо го про-
цесу за відсут ності та в при сут ності білко вих фак торів hRPA і
hPCNA. Вис нов ки. По ка за но, що 1) об ид ва фер мен ти мо жуть ве-
сти TLS на ви ко рис та них ДНК-суб стра тах не за леж но від умов
ре акції, однак ДНК-поліме ра за λ ви я ви ла ся точнішим фермен том;
2) hRPA сти му лює ефек тивність не му та ген но го TLS, ка талізова-
ного ДНК-поліме ра зою λ, без по се ред ньо при вбу до ву ванні нук ле о -
ти ду на впро ти 5-foU і не діє на ефек тивність цьо го про цесу у
тому разі, коли по шкод жен ня зсу ну те в дуп лек сну час ти ну ДНК;
3) hPCNA не впли ває на ефек тивність TLS, ка талізо ва но го об ома
фер мен та ми.
Клю чові сло ва: син тез ДНК че рез по шкод жен ня, ДНК-поліме -
ра зи β и λ, 5-формілу ри дин.
221
BELOUSOVA E. A., LAVRIK O. I.
dNTP
Mg2+ Mn2+
– Replication
factor + hRPA + hPCNA hRPA/hPCNA – Replication
factor + hRPA + hPCNA hRPA/hPCNA
dATP 0.051 0.055 0.049 0.052 12.6 12.9 12.1 12.9
dGTP 0.005 0.006 0.006 0.006 0.31 0.33 0.29 0.35
(kcat/Km)incor/
(kcat/Km)cor*
0.101 0.099 0.119 0.108 0.024 0.026 0.024 0.027
The values shown were obtained under the conditions when replication factors were present at the maximal concentrations; *(kcat/Km)incor/(kcat/Km)cor
values in the presence or absence of replicaton factors. Note: the results are presented as the average value of three independent experiments.
Table 1
The efficacy of dNTP incorporation by pol β (kcat/Km, µM–1s–1)
dNTP – Replication factor + hRPA + hPCNA hRPA/hPCNA – Replication factor** + hRPA**
dATP 0.111 0.108 0.116 0.401 – –
dGTP 0.302 0.008 0.287 0.365 0.137 0.126
(kcat/Km)incor/
(kcat/Km)cor*
2.72 0.08 2.47 0.91 – –
The values shown were obtained under the conditions when replication factors were present at the maximal concentrations; *(kcat/Km)incor/(kcat/Km)cor
values in the presence or absence of replicaton factors; **the efficacy constant values for DNA synthesis using DNA with + 1 primer length. Note:
the results are presented as the average value of three independent experiments. Standard error was estimated as 10 %.
Table 2
The efficacy of dNTP incorporation by pol λ in the presence of Mn2+ ions
Е. А. Бе ло у со ва, О. И. Лав рик
Активность ДНК-по ли ме раз β и λ при син те зе ДНК-суб стра тов, со -
дер жа щих 5-фор ми лу ри дин
Ре зю ме
Цель. Иссле до вать TLS-ак тив ность ДНК-по ли ме раз β и λ че ло ве-
ка на ДНК-дуп лек сах, со дер жа щих 5-фор ми лу ри дин (5-foU) и ими-
ти ру ю щих ин тер ме ди а ты реп ли ка ции ли ди ру ю щей цепи ге ном -
ной ДНК, а так же вли я ние на нее реп ли ка тив ных фак то ров hRPA
и hPCNA. Ме то ды. «За дер жка в геле» (EMSA), фер мен та тив ная
ки не ти ка. Ре зуль та ты. Изу че на спо соб ность ДНК-по ли ме раз β и
λ ка та ли зи ро вать син тез ДНК че рез 5-foU. Опре де ле ны ки не ти-
чес кие ха рак те рис ти ки это го про цес са в от су тствие и в при сут-
ствии бел ко вых фак то ров hRPA и hPCNA. Вы во ды. По ка за но,
что: 1) оба фер мен та спо соб ны вести TLS на ис поль зо ван ных
ДНК-суб стра тах не за ви си мо от усло вий ре ак ции, одна ко ДНК-
по ли ме ра за λ ока за лась бо лее точ ным фер мен том; 2) hRPA сти -
му ли ру ет эф фек тив ность не му та ген но го TLS, ка та ли зи ру е мо го
ДНК-по ли ме ра зой λ, не пос ре дствен но при встра и ва нии нук ле о -
ти да на про тив 5-foU и не вли я ет на эф фек тив ность это го про -
цес са в том слу чае, ког да по вреж де ние сдви ну то в дуп лек сную
часть ДНК; 3) hPCNA не вли я ет на эф фек тив ность TLS, ка та ли -
зи ру е мо го об ои ми фер мен та ми.
Клю че вые сло ва: син тез ДНК че рез по вреж де ние, ДНК-по ли -
меразы β и λ, 5-фор ми лу ри дин.
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SYNTHESIS BY DNA POLYMERASES β AND λ ACROSS 5-foU
|
| id | nasplib_isofts_kiev_ua-123456789-156849 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T16:36:08Z |
| publishDate | 2012 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Belousova, E.A. Lavrik, O.I. 2019-06-19T06:55:43Z 2019-06-19T06:55:43Z 2012 Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates / E.A. Belousova, O.I. Lavrik // Вiopolymers and Cell. — 2012. — Т. 28, № 3. — С. 218–222. — Бібліогр.: 21 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000051 https://nasplib.isofts.kiev.ua/handle/123456789/156849 577.151.45; 57.037; 577.325.5 Цель. Исследовать TLS-активность ДНК-полимераз β и λ человека на ДНК-дуплексах, содержащих 5-формилуридин (5-foU) и имитирующих интермедиаты репликации лидирующей цепи геномной ДНК, а также влияние на нее репликативных факторов hRPA и hPCNA. Методы. «Задержка в геле» (EMSA), ферментативная кинетика. Результаты. Изучена способность ДНК-полимераз β и λ катализировать синтез ДНК через 5-foU. Определены кинетические характеристики этого процесса в отсутствие и в присутствии белковых факторов hRPA и hPCNA. Выводы. Показано, что: 1) оба фермента способны вести TLS на использованных ДНК-субстратах независимо от условий реакции, однако ДНК-полимераза λ оказалась более точным ферментом; 2) hRPA стимулирует эффективность немутагенного TLS, катализируемого ДНК-полимеразой λ, непосредственно при встраивании нуклеотида напротив 5-foU и не влияет на эффективность этого процесса в том случае, когда повреждение сдвинуто в дуплексную часть ДНК; 3) hPCNA не влияет на эффективность TLS, катализируемого обоими ферментами.
 Kлючевые слова: синтез ДНК через повреждение, ДНК-полимеразы β и λ, 5-формилуридин. Мета. Дослідити активність TLS ДНК-полімераз β і λ людини на ДНК-дуплексах, які містять 5-формілуридин (5-foU) та імітують інтермедіати реплікації лідируючого ланцюга геномної ДНК, а також вплив на неї реплікативних факторів hRPA и hPCNA. Методи. «Затримка в гелі» (EMSA), ферментативна кінетика. Результати. Вивчено здатність ДНК-полімераз β і λ активувати синтез ДНК через 5-foU. Визначено кінетичні характеристики цього процесу за відсутності та в присутності білкових факторів hRPA і hPCNA. Висновки. Показано, що 1) обидва ферменти можуть вести TLS на використаних ДНК-субстратах незалежно від умов реакції, однак ДНК-полімераза λ виявилася точнішим ферментом; 2) hRPA стимулює ефективність немутагенного TLS, каталізованого ДНК-полімеразою λ, безпосередньо при вбудовуванні нуклеотиду навпроти 5-foU і не діє на ефективність цього процесу у тому разі, коли пошкодження зсунуте в дуплексну частину ДНК; 3) hPCNA не впливає на ефективність TLS, каталізованого обома ферментами
 Ключові слова: синтез ДНК через пошкодження, ДНК-полімерази β і λ, 5-формілуридин. Aims. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ) using 5-formyluridine (5-foU) containing DNA duplexes which are imitating the intermediates during replication of the leading DNA strand, and to study the influence ofreplication factors hRPA and hPCNA on this activity. Methods. The EMSA and the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis across 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i) both proteins are able to catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate enzyme; (ii) hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol λ at the nucleotide incorporation directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage displaced into the duplex; (iii) hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.
 Keywords: translesion synthesis, DNA polymerases β and λ, 5-formyluridine. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates Активність ДНК-полімераз β і λ при синтезі ДНК-субстратів, які містять 5-формілуридин Активность ДНК-полимераз β и λ при синтезе ДНК-субстратов, содержащих 5-формилуридин Article published earlier |
| spellingShingle | Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates Belousova, E.A. Lavrik, O.I. Structure and Function of Biopolymers |
| title | Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates |
| title_alt | Активність ДНК-полімераз β і λ при синтезі ДНК-субстратів, які містять 5-формілуридин Активность ДНК-полимераз β и λ при синтезе ДНК-субстратов, содержащих 5-формилуридин |
| title_full | Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates |
| title_fullStr | Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates |
| title_full_unstemmed | Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates |
| title_short | Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates |
| title_sort | study of the activity of dna polymerases β and λ using 5-formyluridine containing dna substrates |
| topic | Structure and Function of Biopolymers |
| topic_facet | Structure and Function of Biopolymers |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/156849 |
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