New synthetic substrates of mammalian nucleotide excision repair system

Article abstract of DOI:10.1093/nar/gkt301

DNA probes for the studies of damaged strand excision during the nucleotide excision repair (NER) have been designed using the novel non-nucleosidic phosphoramidite reagents that contain N-[6-(9-antracenylcarbamoyl)hexanoyl]-3- amino-1,2-propandiol (nAnt) and N-[6-(5(6)-fluoresceinylcarbamoyl)hexanoyl]-3- amino-1,2-propandiol (nFlu) moieties. New lesion-imitating adducts being inserted into DNA show good substrate properties in NER process. Modified extended linear nFlu- and nAntr-DNA are suitable for estimation of specific excision activity catalysed with mammalian whole-cell extracts. The following substrate activity range was revealed for the model 137-bp linear double-stranded DNA: nAnt-DNA ≈ nFlu-DNA > Chol-DNA (Chol-DNA - legitimate NER substrate that contains non-nucleoside fragment bearing cholesterol residue). In vitro assay shows that modified DNA can be a useful tool to study NER activity in whole-cell extracts. The developed approach should be of general use for the incorporation of NER-sensitive distortions into model DNAs. The new synthetic extended linear DNA containing bulky non-nucleoside modifications will be useful for NER mechanism study and for applications.

Full text of DOI:10.1093/nar/gkt301

Published online 22 April 2013
Nucleic Acids Research, 2013, Vol. 41, No. 12 e123
doi:10.1093/nar/gkt301
New synthetic substrates of mammalian nucleotide
excision repair system
Alexey Evdokimov1,2, Irina Petruseva1, Aleksandra Tsidulko1,2, Ludmila Koroleva1,2
Inna Serpokrylova1, Vladimir Silnikov1 and Olga Lavrik1,2,
,
*
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of
2
Sciences, Novosibirsk 630090, Russia and Novosibirsk State University, Novosibirsk 630090, Russia
Received July 6, 2012; Revised March 20, 2013; Accepted March 28, 2013
repair investigation is based on the application of syn-
thetic DNA molecules, containing lesion-simulating
adducts. The characterization of specific excision activities
in cell extract and reconstituted systems is a significant
problem and could represent a major concern for funda-
mental research and for medical applications. The avail-
ability of model DNA, which imitates NER substrate well,
is the significant point of NER research progress (2–8).
Methods for design of NER substrate analogues have
been developed. A variety of purine nucleotide derivatives,
such as acetylaminofluorene adducts, products of reac-
tions between nucleobases and benzo[a]pyrene- or
benzo[c]anthracene-diol-epoxides, products of intra-
strand DNA cross-links by cis-dichlorodiamine platinum
(II) and others, have been used for this purpose (9–11).
Py–Py dimers (usually T–T dimers) were used as ana-
logues of damaged pyrimidine bases (12). A number of
investigations were carried out using model DNA contain-
ing the thymine mono-adduct 40-hydroxymethyl-4,50,8-
trimethylpsoralen, or thymidine in which a fluorescein
residue was introduced into C5 position via a linker
fragment (9,13–15). However, synthesis of the model
DNAs, containing damage-mimicking modification of
the defined structure at the desirable position of DNA
molecule, remains up to now actual problem. Studies are
in progress to improve the synthesis and to extend the
variety of model DNA lesions. The elaboration of ‘ultra-
mild’ method of single dG-AAF adduct introduction in
DNA by solid-phase DNA synthesis substantially
facilitated production of oligonucleotides, containing
these modifications (9,16). Fluorescein residue and
several arylazide groups introduced via different linkers
at the nucleobase were successfully applied to imitate
DNA damages produced by the action of aromatic hydro-
carbon derivatives. To synthesize these DNA substrates,
the different methods were used, including the chemo-en-
zymatic approach (2,5,16–19). The improved enzymatic
approach for synthesis of bulky substituted DNA struc-
tures allowed to synthesize the variety of functional
ABSTRACT
DNA probes for the studies of damaged strand
excision during the nucleotide excision repair
(NER) have been designed using the novel non-
nucleosidic phosphoramidite reagents that contain
N-[6-(9-antracenylcarbamoyl)hexanoyl]-3-amino-
1,2-propandiol (nAnt) and N-[6-(5(6)-fluoresceinyl-
carbamoyl)hexanoyl]-3-amino-1,2-propandiol (nFlu)
moieties. New lesion-imitating adducts being
inserted into DNA show good substrate properties
in NER process. Modified extended linear nFlu– and
nAntr–DNA are suitable for estimation of specific
excision activity catalysed with mammalian whole-
cell extracts. The following substrate activity range
was revealed for the model 137-bp linear double-
stranded DNA: nAnt–DNA & nFlu–DNA > Chol–DNA
(Chol–DNA—legitimate
NER
substrate
that
contains non-nucleoside fragment bearing choles-
terol residue). In vitro assay shows that modified
DNA can be a useful tool to study NER activity in
whole-cell extracts. The developed approach
should be of general use for the incorporation of
NER-sensitive distortions into model DNAs. The
new synthetic extended linear DNA containing
bulky non-nucleoside modifications will be useful
for NER mechanism study and for applications.
INTRODUCTION
The DNA nucleotide excision repair (NER) system recog-
nizes and removes a wide variety of structurally diverse
helix-distorting bulky adducts from DNA, mostly
modified nitrogen bases (Pu or Py) or covalently linked
neighbouring bases, and results in the release of short
oligonucleotides (24–32mer) containing the lesion (1).
One of the advanced and upcoming approaches to DNA
*To whom correspondence should be addressed. Tel: +7 383 363 51 95; Fax: +7 383 363 51 53; Email: lavrik@niboch.nsc.ru
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
ß The Author(s) 2013. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial
re-use, please contact journals.permissions@oup.com

e123 Nucleic Acids Research, 2013, Vol. 41, No. 12
PAGE 2 OF 10
analogues of NER substrates, including the photo-
activable ones (20). There is also at least one type of legit-
imate NER substrate containing bulky non-nucleoside
lesions, namely, Chol fragment, introduced by solid-
phase DNA synthesis (21). This type of model DNA has
been used to investigate both UVrABC and eukaryotic
NER systems (22). Unfortunately, most of the NER sub-
strates demonstrate low specific excision efficiency in vitro;
therefore, usage of model DNA for evaluation of NER
system activity is restricted, especially in cell or tissue
extracts (23).
molecular mass measurements. Analytical thin-layer
chromatography (TLC) was performed on Kieselgel 60
F254 pre-coated aluminium plates (Merck); spots
were visualized under ultraviolet light (254 nm). Silica
gel column chromatography was performed using
Merck Kieselgel 60 0.040–0.063 mm. Cholesteryl–TEG–
phosphoramidite (TEG - Dimethoxytrityloxy-3-O-(N-
cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-
(2-cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite) was
from Glen Research. The oligonucleotide synthesis was
performed on
a DNA–RNA synthesizer ASM-800
(Biosset) using standard manufacturer’s protocols. The
regular ONT for creation of model DNA duplexes and
the cholesterol-containing ONT were produced at the la-
boratory of medical chemistry of ICBFM SB RAS (all the
sequences are presented in Supplementary Table S1).
a-[32P]-dCTP and (3000 Ci/mmol) were produced at
ICBFM SB RAS. Taq DNA polymerase, T4 DNA
ligase and T4 polynucleotide kinase were from Biosan
(Novosibirsk), and exonuclease ꢀ was kindly provided
by A. I. Zakabunin (ICBFM SB RAS). Proteinase K
was from Sigma. Dulbecco’s modified Eagle’s medium
(DMEM) and DMEM/F12 mediums for HeLa and
Chinese hamster ovarian (CHO) cells cultivation were
produced by Invitrogen. SiHa and C33A cells biomasses
were generous gift of Natalia and Fedor Kisselev (Institute
of Carcinogenesis, Cancer Research Center, Russian
Academy of Medical Sciences, Moscow).
An effective and easily accessible standard substrate is
necessary to enable the studies of a key stage of NER, the
elimination of the damaged DNA fragment. This will
permit to bring a further insight into the mechanisms of
NER, its role in the development of degenerative diseases
and to investigate the effects of gene–environment inter-
actions on DNA repair.
The main goal of the present investigation was to design
artificial lesions, which can be introduced at any specified
DNA position regardless of nucleotide sequence and effi-
ciently recognized and processed by NER system. This
required three main issues to be resolved: (i) the efficient
preparation of the appropriate phosphoramidites; (ii) the
development of straightforward solid-phase DNA synthe-
sis protocol for the incorporation of the new amidites that
does not compromise further enzymatic integration of the
modified ONT in DNA structures; (iii) the characteriza-
tion of the substrate properties of the model DNA in the
reaction of specific NER excision. Furthermore, it may
serve as an example for the development of additional
functional DNA repair tests, which are needed to gain
further insight into the role of DNA repair in cancer
risk and pathology.
The components for polyacrylamide gel preparation
and the main components of buffer systems were
produced by Sigma.
Synthesis of phosphoramidites
The organic synthesis initial stages
The organic synthesis initial stages are described in detail
in Supplementary Material (see Supplementary Figures
S1–S4 and attached description).
MATERIALS AND METHODS
Reagents and equipment
General procedure
Reagents obtained from commercial suppliers were used
without further purification. Anhydrous solvents were
prepared by distillation under an argon atmosphere in
the presence of appropriate drying agents. DMF was dis-
Compound 5 (7) (Figure 1) (1.05 mmol) was dissolved in a
freshly distilled CH2Cl2 (10 ml), diisopropylammonium
tetrazolide (90 mg, 0.525 mmol) was added, followed by
addition
of
N,N,N0,N0-tetraisopropyl-(2-cyano)ethyl
˚
tilled under reduced pressure and stored over 4 A molecu-
phosphodiamidite (0.50 ml, 1.575 mmol). After 2 h, the
reaction mixture was evaporated, the residue was treated
with hexane (30 ml) and the mixture was kept overnight at
ꢀ20ꢁC. Hexane was then decanted, and the residue was
purified by chromatography on silica gel column with 1%
Et3N in hexane:CH2Cl2 (1:1). Elution with 1% Et3N in
CH2Cl2 gave the target fractions. Fractions containing the
product were combined and evaporated in vacuo. The
residue was then dissolved in dichloromethane (2 ml),
and the product was precipitated with a 10-fold volume
of hexane.
lar sieves under argon. The following reagents were used:
4,40-dimethoxytrityl chloride (Chem-IMPEX, USA), N,N-
ethyldiisopropylamine and pivalic anhydride (DIEA,
Fluka Chemie AG, Switzerland). The 5(6)-carboxy-30,60-
O-dipivaloylfluorescein (24) was prepared as described.
1
All other reagents were from Aldrich (USA). H and 31P
nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker 400 and 300 MHz NMR spectrometers in
deuterated solvents. Chemical shifts are reported in parts
per million (ppm) relative to the tetramethylsilane as the
internal standard. Matrix-assisted laser desorption/ioniza
tion-time of flight (MALDI–TOF) mass spectra were run
on Autoflexspeed or Macroflex spectrometers (Bruker
Daltonics, Germany) in positive-ion mode with
dihydroxybenzoic acid as a matrix. Ion trap XTC ultra
(Agilent Technologies, USA) mass spectrometer with an
electrospray ionization (ESI) interface was also used for
Compound 6
1
Yield 6 74.9%, TLC (CH2Cl2/(CH3)2CO 4:1) Rf 0.74. H
NMR (CDCl3, d) 8.48 [1H, s, Ant-H10], 8.08 [2H, d, J 8.3,
Ant-H1,8], 8.02 [2H, d, J 8.3, Ant-H1,8], 7.57–7.42 [4H, m,
Ant-H2,3,6,7], 7.31–7.19 [9H, m, ArH, DMT], 6.82 [4H,
m, J 8.8, ArH, DMT]; 4.04 [1 H, m, CHOH], 3.77

PAGE 3 OF 10
Nucleic Acids Research, 2013, Vol. 41, No. 12 e123
Figure 1. The general scheme of the organic synthesis procedure. Reagents and conditions: (i) N-hydroxysuccinimide ester of TFA-NH-(CH2)5-
COOH, Et3N, DMF; (ii) DMTCl, pyridine; (iii) aqueous NH3 (25%), pyridine; (iv) (a) 9-anthracenecarboxylic acid, N-hydroxybenzotriazole, DCC,
CH2Cl2, Et3N, (b) 5(6)-carboxyfluorescein-NHS ester, Et3N, CH2Cl2; and (v) 2-cyanoethyl-N,N,N0,N0-tetraisopropylphosphodiamidite, diisopropy-
lammonium tetrazolide, CH2Cl2.
[s, 6 H, OCH3], 3.76–3.04 [10H, m, NCH, POCH2,
NCH2(CH2)4 C(O), NHCH2CH(OH)CH2O], 2.58 (1 H, t,
J 6.1, CH2CN), 2.43 (1 H, t, J 6.3, CH2CN), 2.15 [2H, m,
N(CH2)4CH2C(O)], 1.49 [2H, m, NCH2CH2(CH2)3 C(O)],
1.29 [4H, m, N(CH2)2(CH2)2CH2C(O)], 1.23–1.10 [12H,
m, CH(CH3)2]; 31P NMR (CDCl3, d) 149.38 (s), 148.93
(s); ESI MS (M+Et3N): 1012.5 (calcd 1011.6).
0.01 M iodine in tetrahydrofurane/pyridine/water to
stable P(V) phosphate. Purification was accomplished
using RP-Cartridge (ChemGenes Inc.) according to rec-
ommended methods. Oligonucleotide derivatives were
analysed by polyacrylamide gel electrophoresis (PAGE)
under denaturing conditions and in every case
demonstrated single band purity.
The cholesterol residue attached to linker backbone
instead of a nucleoside was introduced using TEG–
phosphoramidite into the desired sequence by standard
phosphoramidite chemistry at position of the top strand.
Compound 8
1
Yield 8 64.5%, TLC (CH2Cl2/(CH3)2CO 4:1) Rf 0.81. H
NMR (CD3)2CO, d) 8.50 [0.6 H, s, 5-isomer Flu-H4], 8.34
[0.6 H, d, J 8.1, 5-isomer Flu-H6], 8.27 [0.4 H, d, J 8.0,
6-isomer Flu-H5], 8.14 [0.6 H, bt, NH], 8.08 [0.4 H, d, J
8.0, 6-isomer Flu-H4], 7.98 [0.4 H, bt, NH], 7.82 [0.4 H, s,
6-isomer Flu-H7], 7.52–7.18 [12H, m, 5-isomer Flu-H7,
Synthesis of long DNA probes
nAnt- and nFlu-modified oligonucleotides (Figure 2) of
length corresponding to 16mer unmodified ONT were
enzymatically phosphorylated at the 50-ends using T4
polynucleotide kinase and adenosine triphosphate
(ATP). They were ligated with flanking oligonucleotides
(Supplementary Table S1) 1, 2 to yield single-stranded
137mer DNA. Resulting fragments were purified using
PAGE under denaturing conditions. After spectrophoto-
metric quantification, the 137mer ssDNA fragments were
annealed with complementary unmodified strand and
purified using non-denaturing PAGE. In the obtained
duplexes, the internal modification was placed at the
68th position from the 50-ends of the modified 137mer
strand. The cholesterol-containing ONT was used to syn-
thesize 137mer ssDNA for construction of DNA duplex,
which served as positive control of NER activity in mam-
malian cells extracts. As a negative control, 137mer DNA
strands synthesized with unmodified 16mer ONT were
used for duplex formation. The ligation procedure was
described in detail elsewhere (20).
40,50
, ArH, DMT], 7.00–6.83 [8H m, ArH, DMT, Flu-
], 4.11 [1 H, m, CHOH], 3.76 [s, 6 H, OCH3],
10,20,70,80
H
3.69–3.05 [10H, m, NCH, POCH2, NCH2(CH2)4 C(O),
NHCH2CH(OH)CH2O], 2.72 (1 H, bt, CH2CN), 2.62
(1 H, bt, CH2CN), 2.15 [2H, m, N(CH2)4CH2C(O)], 1.60
[2H, m, NCH2CH2(CH2)3 C(O)], 1.34 [18H, s, C(CH3)3],
1.20 [4H, m, N(CH2)2(CH2)2CH2C(O)], 1.11 [12H, m,
CH(CH3)2]; 31P NMR [(CD3)2CO, d] 150.20 (s), 149.82
(s); ESI MS (M+Et3N): 1334.1 (calcd 1333.7).
Oligonucleotide synthesis
Oligonucleotide synthesis was performed using ASM-800
DNA/RNA synthesizer (Biosset) on a 0.1 mM scale.
Standard phosphoramidite chemistry was applied for syn-
thesis (25,26). The procedure consists of cycles of four
chemical reactions: (i) removal of the DMTr-group (4,4’-
Dimethoxytrityl) with 2% dichloroacetic acid in dichloro-
methane; (ii) coupling of phosphoramidite; (iii) capping
with acetic anhydride and 1,4-dimethylaminopyridine;
and (iv) oxidation of P(III) phosphite triester with
Complementary unmodified 137mer ssDNA was
produced using polymerase chain reaction (PCR) and

e123 Nucleic Acids Research, 2013, Vol. 41, No. 12
PAGE 4 OF 10
Figure 2. The non-nucleoside fragments of the modified DNA strand, containing N-[6-(9-antracenylcarbomoyl)hexanoyl]-3-amino-1,2-propandiol
[nAnt, (A)], N-[6-(5(6)-fluoresceinylcarbomoyl)hexanoyl]-3-amino-1,2-propandiol [nFlu, (B)] or cholesterol residue [Chol, (C)].
subsequent treatment of PCR products with exonuclease ꢀ.
The template was pBR322 DNA (positions from 56 to 193
of the upper strand) and primers—50-aattgctaacgcagtcagg-
30 and 50-P-tggacgatatcccgcaagaggc-30 (one of the primers
was 50-phosphorylated). Unmodified 137 nt ssDNA was
produced via treatment of hemiphosphorylated PCR frag-
ments with exonuclease ꢀ. The reaction was performed
under conditions and in the buffer supplied by New
England Biolabs. To produce model dsDNA, the
ssDNA was annealed with a complementary modified
strand. The duplexes were purified in 6% polyacrylamide
gel, 1 ꢂ TBE under non-denaturing conditions to remove
remaining ssDNA.
extract type specified in the legends for figures) in
1 ꢂ NER buffer (25 mM Tris–HCl, pH 7.8, 45 mM
NaCl, 4.4 mM MgCl2, 0.1 mM ethylenediaminetetraacetic
acid and 4 mM ATP) were incubated for 15–60 min at
30ꢁC. Unlike the original method (28), in vitro NER
reaction was performed in the presence of the excess of
template 1 to decrease degradation of the excised oligo-
nucleotide by extract nucleases. After incubation, the
reaction mixture was heated to 95ꢁC and slowly cooled
down to room temperature for annealing of excised oligo-
nucleotide to specific template. The 10 ꢂ Taq-polymerase
buffer (1/10 of reaction mixture volume), Taq polymerase
(5–10 activity units) and a-[32P] dCTP (500–600 Bq) were
added, and the probes were incubated for 5 min at 37ꢁC,
then 1.2 ml of dNTP mixture containing 100 mM dATP,
dGTP, dTTP and 50 mM dCTP was added, and incubation
was continued further for 15 min. The reaction was
stopped by adding proteinase K solution (0.5 ml, 4 mg/
ml). After 60 min of proteinase treatment at 37ꢁC,
reaction mixtures were ethanol precipitated and analysed
by PAGE under denaturing conditions followed by quan-
titative autoradiography (Molecular Imager FX Pro+,
Quantity One software, BioRad). Quantitative analysis
was performed relatively to total radioactivity in each
lane, using control lane signal as baseline. The influence
of CHO extract protein concentration on the intensity of
the excision products was analysed at the initial step, and
the optimal concentration of the extract proteins
(1.6 mg/ml) was selected for the following analysis.
Whole-cell extracts preparation
Human cervical cancer cell lines (SiHa, C33A and HeLa)
and CHO cell line cells were used to prepare whole-cell
extracts as described previously (27). Extracts were ali-
quoted and stored at ꢀ70ꢁC.
In vitro NER assay
The efficiency of removal of the DNA region containing
model lesions was determined using 30-end-labelling (or
‘fill-in’ synthesis) method as described previously (28),
with several modifications. The reaction mixtures contain-
ing 20 nM model DNA duplex, 500 nM template 50-
gggggctcggcaccgtcaccctggatgctgtagg-p-30 (template 1) and
NER-competent cell extract (the amounts of proteins and

PAGE 5 OF 10
Nucleic Acids Research, 2013, Vol. 41, No. 12 e123
linear double-stranded modified DNA (ldsmDNA)
bearing bulky modification in the internal position of
the DNA strand at approximately equal distances from
the ends of the duplex. Despite reported problems in the
usage of linear NER substrates [e.g. NER inhibition with
several cell extracts proteins, non-specific nuclease degrad-
ation and so forth (23,31,32)], modified dsDNA is univer-
sal and is a widely used model for in vitro NER
investigations. Linear NER substrate analogues of the dif-
ferent length were successfully used to estimate correlation
among the damage d DNA structure and repair efficiency,
as well as for evaluation of kinetic and affinity parameters
of NER proteins interaction with DNA substrates (33–
35). Linear NER substrates were used in affinity cross-
linking experiments (2,20,36–38) and in modern prote-
omics (36).
The aim of the present study was to design the new
effective DNA probes for detection of damaged strand
excision in the NER. The optimal strategy of synthesis
and good substrate properties of ldsmDNA demonstrate
their usability for NER activity measurement. The solid-
phase synthesis using bulky modified non-nucleoside
phosphoamidites was suggested as most straightforward
approach for creation of model DNA independently of
the length and nucleotide sequence. Taking into account
the knowledge of the NER recognizable DNA lesions
(1,3,4,30), the fluorescein and antracene derivatives have
been chosen as bulky substituents.
The method of new non-nucleosidic phosphoramidite
reagents synthesis has been elaborated
The synthesis of new non-nucleoside phosphoramidites
and their incorporation into oligodeoxyribonucleotides
are reported herein.
Figure 3. The scheme of NER in vitro assay. Green hexagon represents
a bulky modification; red asterisk, radioactive label; dark blue and grey
ovals, the sequence of specific and non-specific templates, respectively.
Building block 1 was synthesized from commer-
cially available 3-amino-1,2-propanediol, as depicted in
Figure 1. Initial N-hydroxysuccinimide ester of N-TFA-
hexanoic acid (TFA—trifluoroacetic acid) was condensed
with 2 to give diol/3, which was dimetoxytritylated
without separation and purification to yield the
dimetoxytrityl protected diol 4. Universal intermediate 1
was obtained from compound 4 by removal the TFA-pro-
tecting group with NH3.
The control reaction mixtures were processed and
analysed according to the procedure described earlier
in the text. The first type of control mixture contained
non-modified DNA (nm DNA) of the same sequence
instead of modified duplex. The second control contained
the modified DNAs, but the non-specific template 50-
ggggtcaggcaccgtgtatgaaatctaacaat-p-30
(template
2),
On the basis of the non-nucleosidic reagent 1 (Figure 1),
phosphoramidites with fluorophores and other modifica-
tions can be obtained. The synthesis of the compound 1 is
convenient and involves only three steps. The main differ-
ence from the earlier described phosphoramidites contain-
ing 1,2-diol structural type is the distance between the
amino-group and secondary hydroxyl. An amino group
separated from the backbone by the extended linker
should allow easy introduction of the phosphoramidites
with bulky groups into oligonucleotides. Universal
intermediate 1 was converted to derivatives 5 and 7
by its reaction with 9-anthracenecarboxylic acid in
the presence of HOBt (hydroxybenzotriazole) and
DCC (N,N0-dicycloxeyl-carbodiimid) or with 5(6)-
carboxyfluorescein-NHS ester, respectively (Figure 1).
Activated esters were synthesized in situ without add-
itional purification. Compounds 5 and 7 were converted
which is complementary to undamaged segment of DNA
(see Figure 3 for details). DNA incubated in 1 ꢂ NER
buffer in the absence of extract and processed as described
earlier in the text was used as additional control.
RESULTS AND DISCUSSION
Many previous investigations with artificial NER sub-
strates have been performed using circular structures
(phage or plasmid DNA) bearing lesions in the target
position or statistically distributed in the DNA molecule
(8,15,29,30).The primary evaluation of the substrate
properties of recently suggested photoreactive lesions
Fap-dC (-dU) introduced in pBSK II DNA was described
(16). Another type of model substrates for the eukaryotic
NER system is represented by a long (>120 bp length)

e123 Nucleic Acids Research, 2013, Vol. 41, No. 12
PAGE 6 OF 10
Table 1. The sequences and masses of oligodeoxyribonucleotides with
non-nucleosidic modifications (positions in bold indicate insertion of
non-nucleosidic phosphoramidites: M1 – 6, M2 – 8)
noted that the levels of substrate conversion are usually
low for in vitro NER assays (10,11,30). Also, the specific
excision of the damaged fragments from linear DNA–sub-
strate might be additionally decreased because of the
action of several proteins presented in cell extracts (31,32).
Both of most widely used methods of dual incision
products detection are based on 32[P]-label use.
The method of direct detection of NER cleavage was
developed in 1992 (39) and was used to perform an im-
pressive number of investigations (3,40–43). However, this
approach has some disadvantages. First of all, high
amount of 32[P] should be used (27) to produce the sub-
strate DNA of high-specific radioactivity. Moreover, the
process of DNA–substrate synthesis consists of
complicated procedures, which have to be performed
with high-radioactive substances, especially at the initial
stages. Also, the half-life of radioactive substrates is rela-
tively short, and application of synthesized radioactive
DNA for full-scale research is time-limited.
No
Sequence 50!30
Calc. masses Observed masses
(M+H)+
(M+H)+
I-Ant ATCCAGGG-M1-GACGGTG 5129.0
I-Flu ATCCAGGG-M2-GACGGTG 5281.0
II-Ant CACCGTCG-M1-CCTGGAT 5000.0
II-Flu CACCGTCG-M2-CCTGGAT 5152.0
5129.8
5283.7
5002.2
5153.3
to phosphoramidites with N,N,N0,N0-tetraisopropyl-(2-
cyano)ethyl phosphodiamidite. The structures of the com-
1
pounds were confirmed by H, 31P NMR and by high-
resolution mass spectra.
Synthesized phosphoramidites (6 and 8) are readily
soluble in dry acetonitrile and were used for the internal
functionalization of the oligonucleotide chain during
DNA synthesis. The average coupling efficiency of the
modified phosphoramidites at 0.1 M concentration in
dry acetonitrile and 5 min reaction time was found to be
95–97%. Oligonucleotides (ONTs) were assembled,
removed from their supports, de-protected by concen-
trated aqueous ammonia treatment at 60ꢁC overnight
and analysed by reverse-phase high-performance liquid
chromatography and MALDI–TOF MS. Sequences and
properties of the oligodeoxyribonucleotides bearing
various modifications are summarized in Table 1. The
obtained yields were good, and all the molecular masses
corresponded to the expected values within the accuracy
of their measurement.
These problems were mainly solved by the development
of indirect detection of dual incision, catalysed by NER
system. This method was suggested by Wood and col-
32
leagues (28). [P]-label was introduced into 30-ends of
the specific excision products using DNA polymerase,
a-[32P]-dNTP and complementary template ONT. It was
clearly demonstrated that the bands obtained by this
method correspond to the products of specific excision
catalysed by NER system. For now, this method represents
the accepted technique to analyse activity of NER system
and is widely used for a variety of model DNA substrates
(2,16,44,45). Indirect analysis has several advantages in
comparison with direct method of cleavage detection.
First of all, indirect method requires substantially less
amount of radioactivity but shows the superior sensitivity.
In addition, the non-radioactive model DNA substrates
are stable when stored. The scheme of the approach used
in this study is presented in Figure 3.
It was known from our previous study that the whole-
cell extracts may contain non-specific nucleases. Such nu-
cleases effectively digest single-stranded DNA, which is
the main product of the excision reaction. To improve
the original procedure (28), the subsequent modification
was developed here. The excess of template 1 was intro-
duced into reaction mixture before incubation of the
model NER substrates with cell extracts to protect
single-stranded excision products from their digestion by
nucleases of cell extracts. It permits to increase signifi-
cantly the resulting signal of the excision products
(Figure 4A).
The synthesized ONTs (Table 1) contain 15 standard
nucleotides and 1 non-nucleoside fragment (Figure 2)
and have to imitate 16mer ONT.
We have analysed properties of all modified oligo-
nucleotides (Table 1) as substrates of template-dependent
enzyme—T4 polynucleotide ligase. 50-phosphorylated
ONT, containing n-Ant and n-Flu were efficiently incor-
porated in long DNA structures. Good substrate
properties revealed by modified oligonucleotides in the
reaction of enzymatic ligation allowed synthesis of a set
of 137-nt DNA, containing bulky non-nucleoside groups.
After purification (PAGE, 7 M urea) and quantification,
the modified 137mer ssDNA was used for annealing with
complementary DNA to create 137-bp double-stranded
model DNA.
Testing of the model DNA as substrates for in vitro NER
assay. Analysis of the NER reaction in the different cell
extracts
Wild-type CHO cells were used to prepare ‘NER-com-
petent’ extracts (27). Incubation of nFlu– and nAnt–DNA
with proteins of NER-competent extract derived of CHO
cells followed by ‘fill-in’ synthesis resulted in the appear-
ance of the major bands of the radioactive DNA frag-
ments of specific length and sequence (Figure 4B). The
maximal accumulation of the excision products
was detected after incubation of samples for 30 min
(Figure 4C). The appearance of the multiple bands
reflects the existence of different sites of excision.
The 137-bp linear dsDNAs containing bulky modification
in the internal position of the molecule (position 68 from
50-end) have to meet the DNA size and damage-position
requirements to allow assembly of the functional NER
machinery on artificial NER substrates (3). Substrate
properties of nAnt– and nFlu–DNA have been evaluated
using NER-competent extracts of eukaryotic cells. The
excision reaction (1) results in the release of short oligo-
nucleotides (24–32mer) containing the lesion. It should be
As irrelevant nuclease activities are present in the
whole-cell extracts, the specificity of excision reaction of

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Nucleic Acids Research, 2013, Vol. 41, No. 12 e123
Figure 4. The NER dual incision activity of CHO cells extract. The excision products were detected by annealing to the template followed by end-
labelling using a-[32P]-dCTP and Taq DNA polymerase. The reaction products were resolved on a 10% denaturing polyacrylamide gel. (A) The
comparison between original (28) (lanes 1–3) and modified (lanes 4–6) in vitro NER assays. In contrast to the original method the presence of
template 1 in reaction mixture during the incubation of the model DNA substrate with cell extract increases the observed signal intensity. Model
DNAs were incubated for 30 min at 30ꢁC with cell extracts prepared from CHO cells (20 nL DNA, 1.6 mg/ml of extract proteins with or without
500 nM template 1). (B) Specific product accumulation. The 20 nL model DNAs was incubated for 15–60 min at 30ꢁC with 1.6 mg/ml of extract
proteins and 500 nM template 1. (C) Time dependence of the specific excision products accumulation. Grey squares correspond to nFlu–DNA; black
circles, nAnt–DNA. The maximal products accumulation was taken as 100% and was detected for samples incubated for 30 min. The subsequent
incubation leads to non-specific products degradation. (D) Comparison of the results obtained with template 1 and template 2.
bulky lesion from ldsmDNA catalysed by proteins of the
extracts was the subject under particular investigation.
The products of specific excision were not observed
when the control non-modified DNA was incubated
with the extract (Figure 4B, lines 1, 2), or in the case
when ‘non-specific’ template 2 (see Figure 2 for details)
was used in the experiments (Figure 4D). The nFlu– and
nAnt–DNA were evaluated as three to five times more
efficient substrates in comparison with Chol–DNA
(Figure 5). To the best of our knowledge, this is the first
direct comparison of the excision efficiency for Chol-con-
taining DNA with other non-nucleoside substrates.
was found only for linear 137mer Flu-dU–DNA (20).
The products of NER-specific excision in HeLa cell
extract were also not observed using linear 120mer DNA
containing well-defined damage, namely, AAF-dG (9).
AAF-dG was efficiently repaired by NER system in vitro
only when it was introduced into circular DNA (9,15). As
it was aforementioned, the other activities in the cell
extracts, such as a non-specific nucleases and DNA-
binding proteins of high affinity to DNA, particularly
Ku 70–80 antigen, the protein, high content of which is
so typipal for cancer cells extracts, can impede detection of
the NER reaction for ldsmDNA (23,31,32).
Recently the series of 137mer DNAs bearing bulky
modifications as substitutions of nucleobase (including
Flu-dU and Ant-dC) were analysed in vitro NER assay.
The affinity of the model DNA bearing this type of lesion
to NER proteins (46–48) and their capability to be effi-
cient competitors for DNA substrate, containing
benz[a]pyrene residue attached to dG, was found.
However, the excision reaction of the relatively low level
In the present investigation, the new model DNAs were
used to test NER dual incision activity of cancer cells
extracts produced using HeLa, SiHa and C33A human
cancer cell lines. It was observed that NER system of
these cells also efficiently removed the damaged DNA
fragments from nFlu– and nAnt–DNAs (Figure 6).
The results obtained indicate that the new model lesions
imitate efficient NER substrates.

e123 Nucleic Acids Research, 2013, Vol. 41, No. 12
PAGE 8 OF 10
Figure 5. (A) The NER dual incision activity of CHO cells extract. Model DNAs were incubated for 30 min at 30ꢁC with cell extracts prepared from
CHO cells (20 nM model DNA, 0.3 or 1.6 mg/ml of extract proteins and 500 nM template 1).The excision products, containing nFlu, nAnt or Chol,
were detected by annealing to a template followed by end-labelling using a-[32P]-dCTP and Taq DNA polymerase. The reaction products were
resolved on a 10% denaturing polyacrylamide gel. Non-modified 137-bp DNA was used as a negative control; 32P-labelled oligonucleotides were used
as size marker (lane M). (B) Relative signal of the target products for non-modified, nFlu– and nAnt–DNA after incubation with different
concentrations of the extract proteins.
modifications. The method of their incorporation into
DNA and analysis of the obtained model DNAs as sub-
strates of mammalian NER system were developed. The
designed phosphoamidites can be used in the standard
solid-phase synthesis protocol to synthesize similar oligo-
nucleotides of any length and sequence. Mammalian NER
system processes long double-stranded model DNA con-
taining nFlu- and nAnt-lesions with high efficiency in dif-
ferent cell extracts. The possibility to generate modified
DNA containing NER-recognizable lesions using solid-
phase synthesis is a significant advance. It provides unim-
paired access to the well-defined efficient substrates for
NER studies. The additional advantages of the new
DNA derivatives are good substrate properties of the rela-
tively short nAnt- and nFlu-oligonucleotides in the reac-
tions of enzymatic ligation using T4 polynucleotide ligase
and elongation using DNA polymerases). This opens new
perspectives for design of the efficient DNA probes for
affinity cross-linking and for modern proteomic
research. New artificial DNA, containing bulky non-nu-
cleoside modifications will be useful for medical research.
Figure 6. (A) The NER dual incision activity of cancer cells extracts.
SUPPLEMENTARY DATA
Model DNAs were incubated for 30 min at 30ꢁC with cell extracts
prepared from HeLa, C33A or SiHa cells (20 nL DNA, 1.6 mg/ml of
extract proteins and 500 nM template 1). The excision products were
detected by annealing to template followed by end-labelling using
a-[32P]-dCTP and Taq DNA polymerase. The reaction products were
resolved on a 10% denaturing polyacrylamide gel. Non-modified 137-
bp DNA was used as a negative control; 32P-labelled oligonucleotides
were used as size marker. (B) Densitometric analysis of the lanes 5
(HeLa, left), 8 (C33A, middle) and 11 (SiHa, right).
Supplementary Data are available at NAR Online:
Supplementary Table 1 and Supplementary Figures 1–4.
ACKNOWLEDGEMENTS
The authors thank Professor Alexandre Boutorine
(Museum National d’Histoire Naturelle, Paris) for
careful reading of the manuscript and his advices.
SUMMARY AND CONCLUSIONS
FUNDING
We have reported the synthesis of the novel non-
nucleoside phosphoamidites for the solid-phase syn-
thesis of deoxyoligoribonucleotides containing bulky
Russian Fund for Basic Research [RFBR-12-04-00487‘],
[RFBR-12-04-33162]; Russian Ministry of Education and

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Nucleic Acids Research, 2013, Vol. 41, No. 12 e123
damage recognition proteins XPC-HR23B, XPA and RPA to
photoreactive probes that mimic DNA damages. Biochim.
Biophys. Acta, 1770, 781–789.
Science [projects 14.B37.21.0188 and 02.740.11.0079];
Program of Russian Academy of Science on Molecular
and Cellular Biology. Funding for open access charge:
Russian Fund for Basic Research [RFBR-12-04-00487‘];
Program of Russian Academy of Science on Molecular
and Cellular Biology.
17. Khodyreva,S.N. and Lavrik,O.I. (2005) Photoaffinity labeling
technique for studying DNA replication and DNA repair. Curr.
Med. Chem., 12, 641–655.
18. Maltseva,E.A., Rechkunova,N.I., Petruseva,I.O., Silnikov,V.N.,
Vermeulen,W. and Lavrik,O.I. (2006) Interaction of nucleotide
excision repair factors RPA and XPA with DNA containing
bulky photoreactive groups imitating damages. Biochemistry
(Mosc), 71, 270–278.
Conflict of interest statement. None declared
19. Petruseva,I.O., Tikhanovich,I.S., Maltseva,E.A., Safronov,I.V. and
Lavrik,O.I. (2009) Photoactivated DNA analogs of substrates of
the nucleotide excision repair system and their interaction with
proteins of NER-competent HeLa cell extract. Biochemistry
(Mosc), 74, 491–501.
REFERENCES
1. Gillet,L.C. and Scharer,O.D. (2006) Molecular mechanisms of
mammalian global genome nucleotide excision repair. Chem. Rev.,
106, 253–276.
20. Evdokimov,A.N., Petruseva,I.O., Pestryakov,P.E. and Lavrik,O.I.
(2011) Photoactivated DNA analogs of substrates of the
nucleotide excision repair system and their interaction with
proteins of NER-competent extract of HeLa cells. Synthesis and
application of long model DNA. Biochemistry (Mosc), 76,
157–166.
21. Matsunaga,T., Mu,D., Park,C.H., Reardon,J.T. and Sancar,A.
(1995) Human DNA repair excision nuclease. Analysis of the
roles of the subunits involved in dual incisions by using anti-XPG
and anti-ERCC1 antibodies. J. Biol. Chem., 270, 20862–20869.
22. Verhoeven,E.E., Wyman,C., Moolenaar,G.F., Hoeijmakers,J.H.
and Goosen,N. (2001) Architecture of nucleotide excision repair
complexes: DNA is wrapped by UvrB before and after damage
recognition. EMBO J., 20, 601–611.
23. Langie,S.A., Cameron,K.M., Waldron,K.J., Fletcher,K.P., von
Zglinicki,T. and Mathers,J.C. (2011) Measuring DNA repair
incision activity of mouse tissue extracts towards singlet oxygen-
induced DNA damage: a comet-based in vitro repair assay.
Mutagenesis, 26, 461–471.
24. Adamczyk,M., Chan,C.M., Fino,J.R. and Mattingly,P.G. (2000)
Synthesis of 5- and 6-hydroxymethylfluorescein phosphoramidites.
J. Org. Chem., 65, 596–601.
2. Tapias,A., Auriol,J., Forget,D., Enzlin,J.H., Scharer,O.D.,
Coin,F., Coulombe,B. and Egly,J.M. (2004) Ordered
conformational changes in damaged DNA induced by nucleotide
excision repair factors. J. Biol. Chem., 279, 19074–19083.
3. Huang,J.C. and Sancar,A. (1994) Determination of minimum
substrate size for human excinuclease. J. Biol. Chem., 269,
19034–19040.
4. Geacintov,N.E., Broyde,S., Buterin,T., Naegeli,H., Wu,M., Yan,S.
and Patel,D.J. (2002) Thermodynamic and structural factors in
the removal of bulky DNA adducts by the nucleotide excision
repair machinery. Biopolymers, 65, 202–210.
5. DellaVecchia,M.J., Croteau,D.L., Skorvaga,M., Dezhurov,S.V.,
Lavrik,O.I. and Van Houten,B. (2004) Analyzing the handoff of
DNA from UvrA to UvrB utilizing DNA-protein photoaffinity
labeling. J. Biol. Chem., 279, 45245–45256.
6. Hermanson-Miller,I.L. and Turchi,J.J. (2002) Strand-specific
binding of RPA and XPA to damaged duplex DNA.
Biochemistry, 41, 2402–2408.
7. Dip,R., Camenisch,U. and Naegeli,H. (2004) Mechanisms of
DNA damage recognition and strand discrimination in human
nucleotide excision repair. DNA Repair (Amst), 3, 1409–1423.
8. Sugasawa,K., Okamoto,T., Shimizu,Y., Masutani,C., Iwai,S. and
Hanaoka,F. (2001) A multistep damage recognition mechanism
for global genomic nucleotide excision repair. Genes Dev., 15,
507–521.
9. Gillet,L.C., Alzeer,J. and Scharer,O.D. (2005) Site-specific
incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene
(dG-AAF) into oligonucleotides using modified ‘ultra-mild’ DNA
synthesis. Nucleic Acids Res., 33, 1961–1969.
25. Agrawal,S. (1993) Protocols for Oligonucleotides and Analogs:
Synthesis and Properties in Methods in Molecular Biology.
Humana Press, Totowa, NJ.
26. Gait,M. (ed.), (1984) Oligonucleotide Synthesis. A Practical
Approach. Oxford, IRL Press Lim.
27. Reardon,J.T. and Sancar,A. (2006) Purification and
characterization of Escherichia coli and human
nucleotide excision repair enzyme systems. Methods Enzymol.,
408, 189–213.
10. Kropachev,K., Kolbanovskii,M., Cai,Y., Rodriguez,F.,
Kolbanovskii,A., Liu,Y., Zhang,L., Amin,S., Patel,D., Broyde,S.
et al. (2009) 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., 386, 1193–1203.
11. Mocquet,V., Kropachev,K., Kolbanovskiy,M., Kolbanovskiy,A.,
Tapias,A., Cai,Y., Broyde,S., Geacintov,N.E. and Egly,J.M.
(2007) The human DNA repair factor XPC-HR23B distinguishes
stereoisomeric benzo[a]pyrenyl-DNA lesions. EMBO J., 26,
2923–2932.
12. Reardon,J.T. and Sancar,A. (2003) Recognition and repair of the
cyclobutane thymine dimer, a major cause of skin cancers, by the
human excision nuclease. Genes Dev., 17, 2539–2551.
13. Mu,D., Tursun,M., Duckett,D.R., Drummond,J.T., Modrich,P.
and Sancar,A. (1997) Recognition and repair of compound DNA
lesions (base damage and mismatch) by human mismatch repair
and excision repair systems. Mol. Cell. Biol., 17, 760–769.
14. Khan,Q.A., Kohlhagen,G., Marshall,R., Austin,C.A.,
Kalena,G.P., Kroth,H., Sayer,J.M., Jerina,D.M. and Pommier,Y.
(2003) Position-specific trapping of topoisomerase II by
benzo[a]pyrene diol epoxide adducts: implications for interactions
with intercalating anticancer agents. Proc. Natl Acad. Sci. USA,
100, 12498–12503.
28. Araujo,S.J., Tirode,F., Coin,F., Pospiech,H., Syvaoja,J.E.,
Stucki,M., Hubscher,U., Egly,J.M. and Wood,R.D. (2000)
Nucleotide excision repair of DNA with recombinant human
proteins: definition of the minimal set of factors, active forms of
TFIIH, and modulation by CAK. Genes Dev., 14, 349–359.
29. Hess,M.T., Gunz,D. and Naegeli,H. (1996) A repair competition
assay to assess recognition by human nucleotide excision repair.
Nucleic Acids Res., 24, 824–828.
30. Buterin,T., Hess,M.T., Gunz,D., Geacintov,N.E., Mullenders,L.H.
and Naegeli,H. (2002) Trapping of DNA nucleotide excision
repair factors by nonrepairable carcinogen adducts. Cancer Res.,
62, 4229–4235.
31. Huang,J.C., Zamble,D.B., Reardon,J.T., Lippard,S.J. and
Sancar,A. (1994) HMG-domain proteins specifically inhibit the
repair of the major DNA adduct of the anticancer drug cisplatin
by human excision nuclease. Proc. Natl Acad. Sci. USA, 91,
10394–10398.
32. Calsou,P., Frit,P. and Salles,B. (1996) Double strand breaks in
DNA inhibit nucleotide excision repair in vitro. J. Biol. Chem.,
271, 27601–27607.
33. Trego,K.S. and Turchi,J.J. (2006) Pre-steady-state binding of
damaged DNA by XPC-hHR23B reveals a kinetic mechanism for
damage discrimination. Biochemistry, 45, 1961–1969.
34. Hey,T., Lipps,G., Sugasawa,K., Iwai,S., Hanaoka,F. and
Krauss,G. (2002) The XPC-HR23B complex displays high affinity
and specificity for damaged DNA in a true-equilibrium
fluorescence assay. Biochemistry, 41, 6583–6587.
15. Alzeer,J. and Scharer,O.D. (2006) A modified thymine for the
synthesis of site-specific thymine-guanine DNA interstrand
crosslinks. Nucleic Acids Res., 34, 4458–4466.
16. Maltseva,E.A., Rechkunova,N.I., Gillet,L.C., Petruseva,I.O.,
Scharer,O.D. and Lavrik,O.I. (2007) Crosslinking of the NER

e123 Nucleic Acids Research, 2013, Vol. 41, No. 12
PAGE 10 OF 10
35. Hey,T., Lipps,G. and Krauss,G. (2001) Binding of XPA and
RPA to damaged DNA investigated by fluorescence anisotropy.
Biochemistry, 40, 2901–2910.
36. Guggenheim,E.R., Xu,D., Zhang,C.X., Chang,P.V. and
Lippard,S.J. (2009) Photoaffinity isolation and identification of
proteins in cancer cell extracts that bind to platinum-modified
DNA. Chembiochem, 10, 141–157.
37. Khodyreva,S.N., Prasad,R., Ilina,E.S., Sukhanova,M.V.,
Kutuzov,M.M., Liu,Y., Hou,E.W., Wilson,S.H. and Lavrik,O.I.
Apurinic/apyrimidinic (AP) site recognition by the 50-dRP/AP
lyase in poly(ADP-ribose) polymerase-1 (PARP-1). Proc. Natl
Acad. Sci. USA, 107, 22090–22095.
38. Sukhanova,M.V., D’Herin,C., van der Kemp,P.A., Koval,V.V.,
Boiteux,S. and Lavrik,O.I. (2011) Ddc1 checkpoint protein and
DNA polymerase varepsilon interact with nick-containing DNA
repair intermediate in cell free extracts of Saccharomyces
cerevisiae. DNA Repair (Amst), 10, 815–825.
42. Ding,S., Kropachev,K., Cai,Y., Kolbanovskiy,M.,
Durandina,S.A., Liu,Z., Shafirovich,V., Broyde,S. and
Geacintov,N.E. Structural, energetic and dynamic properties of
guanine(C8)-thymine(N3) cross-links in DNA provide insights on
susceptibility to nucleotide excision repair. Nucleic Acids Res., 40,
2506–2517.
43. Buterin,T., Meyer,C., Giese,B. and Naegeli,H. (2005) DNA
quality control by conformational readout on the undamaged
strand of the double helix. Chem. Biol., 12, 913–922.
44. Staresincic,L., Fagbemi,A.F., Enzlin,J.H., Gourdin,A.M.,
Wijgers,N., Dunand-Sauthier,I., Giglia-Mari,G., Clarkson,S.G.,
Vermeulen,W. and Scharer,O.D. (2009) Coordination of dual
incision and repair synthesis in human nucleotide excision repair.
EMBO J., 28, 1111–1120.
45. Fagbemi,A.F., Orelli,B. and Scharer,O.D. (2012) Regulation of
endonuclease activity in human nucleotide excision repair. DNA
Repair (Amst), 10, 722–729.
39. Huang,J.C., Svoboda,D.L., Reardon,J.T. and Sancar,A. (1992)
Human nucleotide excision nuclease removes thymine dimers from
DNA by incising the 22nd phosphodiester bond 50 and the 6th
phosphodiester bond 30 to the photodimer. Proc. Natl Acad. Sci.
USA, 89, 3664–3668.
40. Jansen,J., Olsen,A.K., Wiger,R., Naegeli,H., de Boer,P., van Der
Hoeven,F., Holme,J.A., Brunborg,G. and Mullenders,L. (2001)
Nucleotide excision repair in rat male germ cells: low level of
repair in intact cells contrasts with high dual incision activity
in vitro. Nucleic Acids Res., 29, 1791–1800.
46. Petruseva,I.O., Tikhanovich,I.S., Chelobanov,B.P. and Lavrik,O.I.
(2008) RPA repair recognition of DNA containing pyrimidines
bearing bulky adducts. J. Mol. Recognit., 21, 154–162.
47. Krasikova,Y.S., Rechkunova,N.I., Maltseva,E.A., Petruseva,I.O.
and Lavrik,O.I. (2011) Localization of xeroderma pigmentosum
group A protein and replication protein A on damaged DNA in
nucleotide excision repair. Nucleic Acids Res., 38, 8083–8094.
48. Krasikova,Y.S., Rechkunova,N.I., Maltseva,E.A., Petruseva,I.O.,
Silnikov,V.N., Zatsepin,T.S., Oretskaya,T.S., Scharer,O.D. and
Lavrik,O.I. (2008) Interaction of nucleotide excision repair factors
XPC-HR23B, XPA, and RPA with damaged DNA. Biochemistry
(Mosc), 73, 886–896.
41. Sugasawa,K., Akagi,J., Nishi,R., Iwai,S. and Hanaoka,F. (2009)
Two-step recognition of DNA damage for mammalian nucleotide
excision repair: Directional binding of the XPC complex and
DNA strand scanning. Mol. Cell, 36, 642–653.