Photochemistry and Photobiology, 2020, 96: 440–449
Invited Review in Memory of Professor Alain Favre
Photoreactive DNA as a Tool to Study Replication Protein A Functioning
in DNA Replication and Repair
Nadejda I. Rechkunova1,2 and Olga I. Lavrik1,2*
1
Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
2
Department of Natural Sciences, Novosibirsk State University, Novosibirsk, Russia
Received 27 October 2019, accepted 8 December 2019, DOI: 10.1111/php.13222
ABSTRACT
Replication protein A (RPA), eukaryotic single-stranded
DNA-binding protein, is a key player in multiple processes of
DNA metabolism including DNA replication, recombination
and DNA repair. Human RPA composed of subunits of 70-,
32- and 14-kDa binds ssDNA with high affinity and interacts
specifically with multiple proteins. The RPA heterotrimer
binds ssDNA in several modes, with occlusion lengths of 8–
10, 13–22 and 30 nucleotides corresponding to global, transi-
tional and elongated conformations of protein. Varying the
structure of photoreactive DNA, the intermediates of differ-
ent stages of DNA replication or DNA repair were designed
and applied to identify positioning of the RPA subunits on
the specific DNA structures. Using this approach, RPA inter-
actions with various types of DNA structures attributed to
replication and DNA repair intermediates were examined.
This review is dedicated to blessed memory of Prof. Alain
Favre who contributed to the development of photoreactive
nucleotide derivatives and their application for the study of
protein–nucleic acids interactions.
INTRODUCTION
Photocrosslinking is a useful technique to study interaction of
protein and protein complexes with nucleic acids. The method of
affinity modification with the use of reactive DNA structures is
one of the most informative approaches to study dynamic pro-
tein–DNA systems, because it allows crosslinking even unstable
intermediate complexes which play important roles in the
dynamic molecular machines carrying key cellular processes, for
example, DNA replication, transcription and repair (1,2). Pho-
toreactive dNMP (NMP) residues can be introduced into DNA in
the course of DNA transactions by the activity of DNA (or
RNA) polymerases to design photoreactive intermediates of the
process under investigation (1,3). Alternatively, photoreactive
groups can be introduced into DNA via solid-phase synthesis of
oligonucleotides. The various photoreactive groups were syn-
thetized and introduced into nucleotide moieties to provide varia-
tions of the efficiency of photocrosslinking. Wide range of
photoreactive base-substituted derivatives of dNTPs and
*Corresponding author email: lavrik@niboch.nsc.ru (Olga Lavrik)
© 2020 American Society for Photobiology
440
oligonucleotides has been synthesized and used to design pho-
toreactive intermediates of DNA metabolism pathways (1).
Human RPA is a heterotrimeric protein, composed of 70-, 32-
and 14-kDa subunits which harbor six oligonucleotide/oligosac-
charide binding folds (OB-folds; indicated A through F). The
DNA-binding OB-folds have been referred as DNA-binding
domains (DBDs). The functions of this protein are based on its
DNA-binding activity and specific protein–protein interactions.
The major ssDNA-binding activity of RPA is associated with
p70 (4). This subunit contains fore DNA-binding domains: two
central copies of ssDNA-binding motifs (domains A and B) and
a putative zinc finger motif (domain C). The central DNA-bind-
ing domains are necessary and sufficient for interactions with
ssDNA; however, domain C and part of the N-terminus of the
p70 (domain F) are needed for optimal ssDNA-binding activity
(5,6). The fifth DNA-binding domain of RPA, domain D, resides
in the p32 subunit (7). RPA binds ssDNA with a defined polar-
ity: In the crystal structure ssDNA binds to the A and B domains
in a 50 ?30 direction where the 50 -end associated with domain A
and the 30 -end associated with domain B (8). The third RPA sub-
unit, p14, also contains OB domain (E) (9) which possesses
weak DNA-binding activity (10) and is most likely involved in
the formation of a RPA heterotrimer (9).
The three RPA subunits form a stable complex; trimerization
is mediated by the p70C, p32D and p14E domains, which
together form the trimerization core (11). Flexible linkers connect
all other RPA regions to the trimerization core, providing RPA
with high conformational mobility and the capability of using a
broad range of DNA sequences as ligands (12).
It is now accepted that the mechanism of ssDNA binding by
RPA includes sequential binding events (11). First, binding ini-
tially involves an unstable recognition site of 8–10 nt with the
high-affinity DBD A and DBD B domains on the 50 -side of the
occluded ssDNA; it is designated “compact (or globular) confor-
mation” or 8–10 nt binding mode. Second, this step is followed
by the weaker binding of DBD C, on the 30 -side, leading to an
intermediate or “elongated contracted” or “transient” (13–22 nt)
binding mode (13,14). Finally, binding of DBD D on the 30 -side
forms a stable “elongated extended” complex characterized by a
30 nt long occluded binding site (30 nt binding mode). Electron
microscopy data have shown that conformational changes of
RPA accompanied binding process (15,16). When ssDNA binds
to domains A and B, the effective concentration of DNA is

raised so that the lower affinity sites of domains C and D can
bind (17). Recent studies have suggested that the RPA-ssDNA
complex is relatively dynamic where not all DBDs are stably
bound to the DNA and microscopic dissociation of individual
DNA-binding domains occurs (18–21). Thus, although RPA
binds to ssDNA with very high affinity (20,22–24), it can be dis-
placed by DNA-binding proteins with much lower DNA-binding
affinity. This fact is important for RPA functioning in the
dynamic cell systems.
The mechanism of RPA interaction with DNA is investigating
during several decades by different approaches. This review
focuses on advances of photoaffinity labeling technique in detect-
ing the RPA binding modes described above and in proving the
polarity of ssDNA binding.
STRUCTURE AND PHOTOCHEMICAL
CHARACTERISTICS OF PHOTOREACTIVE
DNTP ANALOGUES, SYNTHESIS OF
PHOTOREACTIVE DNA
The most important characteristic of photoreactive groups used
to modify biological molecules is their absorption spectrum, that
is, their ability to be excited by near ultraviolet light, to avoid
stimulating the inherent photoreactivity of nucleic acids and pro-
teins, and to avoid degradation and inactivation caused by ultra-
violet light. Another property of photoreagents is efficiency of
crosslinking to target molecules. Azido group modified nucleo-
tides can be activated at wavelengths >300 nm and produce the
highly reactive nitrene which covalently bound to molecules in
close proximity. Therefore, azido derivatives of nucleotides, in
particular aryl-azido substituted, are widely used for study of the
protein–nucleic acid interactions.
A set of base-substituted dNTP analogues containing different
aryl-azido groups was synthetized and used as the substrates of
DNA polymerases to produce photoreactive DNA for following
photoaffinity modification of DNA polymerases and other DNA-
binding proteins, for example, RPA. Structural formulas, desig-
nations and full names of the dNTP derivatives are shown in
Fig. 1.
Substituents in the aromatic ring effect on the photochemical
characteristics of a reagent and its biochemical features. Substrate
properties of dCTP (Fig. 1a) and dUTP (Fig. 1b) analogues in
primer elongation catalyzed by DNA polymerases and efficiency
of biopolymer modification were intensively analyzed (25–31).
Among these compounds, the dCTP analogue substituted at the
exo-amino group in the 4th position of the pyrimidine ring with a
FAP group (FAP-dCTP on Fig. 1a) provided the combination of
good substrate properties in DNA synthesis catalyzed by DNA
polymerases with effective crosslinking to proteins when intro-
duced into DNA (31). This analogue was used for protein modi-
fication both in reconstituted systems and in cellular extracts
(32–37).
Aryl-azido substituents are large and their attachment to the
base via linker changes a size of nucleotide residue and can
induce distortion in DNA structure and influence on the protein
affinity to DNA. Furthermore, in this case it is unclear whether
crosslinked protein locates at the indicated position of DNA or at
adjacent site and contacts photoreactive group which is available
due to the extended linker. This problem may be solved by using
DNA bearing zero-length crosslinker, such as halopyrimidines
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Photochemistry and Photobiology, 2020, 96 441
(Fig. 2). The substitution of 5-bromo-20 -deoxyuridine (BrdU) for
thymidine in DNA has been used for long time to investigate
DNA-protein interactions via photocrosslinking because excita-
tion can be achieved using ultraviolet light >300 nm (38), and
the Van der Waals radius of bromine (1.95 A) is similar to the
radius of a methyl group (2.0 A), making bromouracil compara-
ble to thymine in size. As a consequence, BrdU-substituted
DNAs show specific binding to proteins with affinities similar to
those of the nonsubstituted DNA. Although the iodine atom of
5-iodo-20 -deoxyuridine (IdU) has slightly larger van der Waals
radius (2.15 A) than the corresponding methyl group, it only
marginally affects nucleic acid structure and protein binding.
Furthermore, IdU possesses more intense absorption at longer
wavelengths than BrdU that allows reducing unwanted photo-
damage from excitation of other light-absorbing chromophores in
the DNA-protein complex. For this reason, IdU has become
more popular in experiments on crosslinking studies of DNA-
protein interactions. It should be noted that both BrdU and IdU
undergo crosslinking reactions only with amino acid side chains
that are in close contact to the photoreactive nucleobase and
exhibit a strong preference for aromatic side chains (39).
Another zero-length chromophore to study nucleic acid-pro-
tein and nucleic acid-nucleic acid interactions is 4-thiouridine (4-
SU). Its structure is similar to uridine, with only the 4-keto oxy-
gen replaced by a sulfur atom (Fig. 2). In comparing the Van
der Waals radii, the sulfur is only 0.45 A larger than oxygen
(40). This small impact in structure variation and high photoreac-
tivity with amino acid residues and nucleic bases make 4-thioura-
cil an excellent chromophore to study nucleic acid-protein and
nucleic acid-nucleic acid interactions. In addition, the substitution
with sulfur at the 4-position centers the main absorption band
around 330 nm, compared with uracil’s absorption band centered
at 260 nm (40). The ability to excite 4-SU at wavelengths above
300 nm allows minimizing unwanted additional photochemistry
or photodamage to the system. The photoreaction of this moiety
in the structure of tRNA was discovered in 1969 by Alain Favre
(41,42). For more than 40 years, Dr. Favre deeply contributed to
the study of 4-SU photochemistry, biochemical properties and its
application for analyzes of the structures and functioning of pro-
tein–nucleic acid complexes. In particular, this photoreactive
probe was used to study interactions between tRNA and aminoa-
cyl-tRNA synthetases (43,44), protein–nucleic acids and nucleic
acids-nucleic acids interactions within human ribosome (45,46),
as well as interaction of RPA with ssDNA (47–49).
MODES OF RPA BINDING TO DNA
Photoreactive DNAs were applied to reveal the arrangement of
RPA subunits on different DNA structures. Initially, using partial
DNA duplex with the 19 nt single-stranded extension and pho-
toreactive dUMP analogue at the 30 -end of the primer, preferable
labeling the p32 subunit was demonstrated (29). In contrast, only
limited crosslinking of p70 and no crosslinking of p14 were
observed. Interaction of the p32 subunit of RPA with nascent
DNA in replicating SV40 chromosomes has also been observed
indicating that the p32 contacts early intermediates produced by
DNA polymerase a-primase (50,51). Further, using several tem-
plate-primer systems differing in the size of the single-stranded
template tail (31 to 4 nt), it was shown that the pattern of p32
and p70 RPA subunit labeling, and consequently their interaction

442 Nadejda I. Rechkunova and Olga I. Lavrik
Figure 1. Aryl-azido substituted analogues of dCTP (a) and dUTP (b). Top—overall structures of the substituted dNTPs, where R1 indicates substitutes
attached to the exo-amino group in the 4th position of the cytosine, R2—substitutes at the 5N-position of the uracyl. Below—structural formulas of R1
and R2, designations and full names of the analogues.
with the template-primer junction, is strongly dependent on the
template extension length at a particular RPA concentration
(13,47,52), as well as on the ratio of RPA to DNA template con-
centration (13). When template extension was shortened up to 14
nt or less, the p70 subunit of RPA was predominantly pho-
tocrosslinked to the 30 -end of the photoreactive primer (13). A
model of changes in the RPA configuration modulated by the
length of the template extension in the course of nascent DNA
synthesis in the replication fork was suggested (Fig. 3a). These
data approved the changes of RPA structure depending on the
length of ssDNA template.
To examine RPA subunit arrangements when bound to the
ssDNA in gap structures, two sizes of single-stranded DNA plat-
forms, 9 and 30 nt, flanked with the photoreactive group at the
30 or 50 margin were used (53,54). The ability of RPA subunits
to be crosslinked to the 30 or 50 photoreactive DNAs indicates
their location in the close proximity to the 30 - or 50 -end of the
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gap in the RPA–DNA complex. The pattern of RPA subunit
crosslinking strongly depended on the location of the photoreac-
tive group and on the gap size (Fig. 3b). When bound to a 9 nt
single-stranded part of gap using the high affinity A and B
DNA-binding domains of p70, RPA adopts the compact confor-
mation in which the p32 subunit crosslinking to the 30 -end of
upstream oligonucleotide is much less in comparison with
crosslinking of p70 subunit. In that case, p70 can be crosslinked
to the both 30 - and 50 -ends of DNA; therefore, suggesting that
p70 is located near both ends of gapped DNA in globular protein
conformation. When RPA binds in the stable elongated confor-
mation, 30 nt of ssDNA is occluded and p70 is located near the
50 -end of oligonucleotide providing downstream orientation of
p32 near the 30 -end of the upstream oligonucleotide along with
the polarity of RPA binding to extended ssDNA platform. The
same subunit orientation can be realized when RPA binds DNA
in the stable elongated conformation when 30 nt of ssDNA is

Figure 2. Structures of zero-length photoreactive crosslinker.
a b
8-10 nt binding mode
globular conformation
13-22 nt binding mode
transient conformation
30 nt binding mode
elongated conformation
Figure 3. RPA binding to ssDNA platform in the partial DNA duplexes
with the 50 -protruding strand (a) and a gap (b). Contacts with the 30 -end
of the primer are indicated by the dashed red circle. The RPA structure
adopts the globular, transient and elongated conformation according to
the size of the ssDNA platform.
occluded by RPA in the case of extended DNA duplex (Fig. 3a).
The data demonstrate that p14 is far from 30 - or 50 -ends of the
DNA gap or masked by other subunits. In addition because RPA
can be crosslinked to the 30 -end of primer under some conditions
these studies suggest that p70 is located in close proximity to
both ends of the gap, at least transiently. It is interesting that the
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Photochemistry and Photobiology, 2020, 96 443
same modes of RPA binding were demonstrated by using pho-
toreactive aryl-azido groups attached via extended linkers to
nucleotide moiety or with the zero length 4-SU crosslinker differ
in photoreactivity (47).
In summary, the pattern of RPA subunit labeling in DNA
structures with gap or template extension is determined by the
polar RPA binding to ssDNA (55) when p70 subunit initially
resides at the end of ssDNA platform and then extends in the
direction of the 30 -end. Subunit orientation near the 30 -end of
DNA structures is predetermined by the size of ssDNA platform
that modulates changes in RPA conformation which appears to
be nearly the same with gap and template extension. Polarity of
RPA binding in elongated conformation has been suggested to
be realized in nucleotide excision repair (NER) for positioning of
the excision nucleases XPG and EXCC1-XPF on the DNA (56).
However, it should be noted that 30 nt binding mode can be
realized only in the case of low ratio of RPA protein concentra-
tion to DNA. Otherwise, RPA binding modes with (8-10) and
(13-22) nt binding sites contribute to cooperative binding several
RPA molecules along ssDNA. It is these low-affinity interactions
can play important role in dynamic processes of DNA replication
and repair (57).
The same DNA structures, namely, partial DNA duplexes
with the 3’- or 5’-recessed photoreactive end or DNA duplexes
with 30 nt gap containing the photoreactive group at either the
3’ or 5’ terminus, were used to analyze an interaction of replica-
tion factor C (RFC) with DNA intermediates of replication or
repair. RFC, a multiprotein complex composed of five subunits,

444 Nadejda I. Rechkunova and Olga I. Lavrik
RFC140, RFC40, RFC38, RFC37 and RFC36, named according
to their molecular masses, is the loader of sliding clamp factor,
proliferating cell nuclear antigen (PCNA), which is essential for
processive synthesis of DNA by eukaryotic DNA polymerases d
and e (58–60). DNA primers containing a photoreactive group at
their 5’-end were able to crosslink with the largest RFC subunit,
RFC140, on both primer-templates and DNA gap structures,
whereas 3’-end photoreactive primers crosslinked only with
RFC140 within the DNA gap structure (61). Addition of RPA to
the reaction mixture resulted in the crosslinking of RPA subunits
and inhibited crosslinking of RFC140 using 3’- but not 5’-pho-
toreactive primers at the gap. PCNA was neither crosslinked with
any DNA used nor influenced on the RFC140 labeling.
Crosslinking of RFC140 to primers with 5’-end photoreactive
group suggests the interaction of RFC with the 5’-end of a nas-
cent DNA primer during DNA replication. This interaction may
be important for the switch of DNA replication from initiation to
elongation. A model for the DNA polymerase switch during
eukaryotic DNA replication was proposed on the basis of these
data (61).
Modification of both the 70- and 32-kDa subunits by pho-
toaffinity labeling indicates that RPA can bind the primer-tem-
plate junction of partial duplex DNAs by interacting with the 30 -
end of the primer. The following experiments were aimed to
investigate conformational changes accompanying the RPA bind-
ing to partial DNA duplexes and assigned protein domains play-
ing important roles in such interactions. Limited proteolysis
combined with immunoblotting identification of the proteolytic
fragments was used to address whether the binding of RPA to
partial DNA duplexes containing 50 -protruding ends shows some
peculiarities in comparison to the ssDNA which binding leads to
a significant conformational changes in heterotrimer (62). It was
shown that the conformation of the RPA bound to partial DNA
duplexes significantly differs from that of RPA bound to ssDNA
of comparable length and of free RPA in solution (63). Prote-
olytic digestion of RPA crosslinked to photoreactive 30 -end of
the primer followed with immunoblotting allow determining that
domains located in the central part of the RPA32 subunit (amino
acids 39–180) and the C-terminal part of the RPA70 subunit
(amino acids 432–616) are involved in interactions with the 30 -
end of the primer in partial DNA duplexes (63). These data are
consistent with the interpretation that RPA binds the primer–tem-
plate junction via contacts with the RPA trimerization core. Fur-
thermore, using crosslinking experiments with RPA deletion
mutants, p70ABCp32Dp14, p70AB, p70Cp32Dp14 (trimeriza-
tion core) and p32Dp14, it was shown that in the absence of
two major DBDs, p70A and p70B, the RPA trimerization core
was able to recognize correctly the primer–template junction and
adopt an orientation similar to that in native RPA (64). In con-
trast, the RPA dimerization core, consisting of p32Dand p14,
was not detectably crosslinked with such DNA structures. There-
fore, p70 seems to be the predominant subunit to bind single-
stranded DNA and this interaction positions the p32 subunit to
the 3’-end of the primer (52). Dimeric RPA complex p70p32
lacking p14 was able to bind single-stranded DNA, but its bind-
ing mode and affinity differed from those of the heterotrimeric
complex. Moreover, in this complex p32 only minimally recog-
nized the 3’-end of a primer in a primer–template junction (65).
These data speak in favor of key role of the RPA trimerization
core in proper orientation of RPA relatively to primer–template
junction in the course of DNA replication.
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As it was mentioned above, no crosslinking of the small sub-
unit of RPA, p14, to aryl-azido-containing DNA was observed.
However, when 30 nt oligonucleotides containing 4-thiodeoxyur-
idine in different positions as zero-length photoreagent were
used, covalent adducts which can be attributed to p14 cross-
linked to such DNA were observed (66). Indeed, further pho-
toaffinity labeling experiments using 31 nt oligonucleotides with
the 4-thiothymine residues introduced at a defined site coupled
with the identification of crosslinked targets using specific anti-
bodies to certain RPA subunits, revealed that in the elongated
extended RPA conformation p14 closely contacts the 30 -end
positioned nucleotides and yields a covalent adduct with 4-SdT
(49). The proposed scheme of elongated complex was in good
agreement with sequential 50 ?30 binding of RPA where the first
DBDs A and B located to the 50 -side start to interact with the
DNA, followed along the DNA by DBD C and DBD D, and
finally DBD E from p14 RPA binds to the DNA progressing to
the 30 nt high-affinity binding mode (Fig. 4). Thus, 4-thio-
thymidine containing DNA can be useful to determine RPA sub-
units positioning on the certain sites of ssDNA. Furthermore,
such DNA probes can be used to trap single-stranded DNA-bind-
ing proteins in cellular extracts, which provides in combination
with mass spectrometry a powerful tool for proteomic investiga-
tions (67).
DNA structures containing single-stranded platforms are com-
mon intermediates of cellular processes of DNA metabolism and
also occur in some specific regions of the genomic DNA. One of
the specific types of ssDNA is a part of telomeric DNA which
locates at the ends of each chromosome and contains G-rich ter-
mini as relatively short single-stranded 30 overhangs designated
G-overhangs (68). RPA was shown to be present at the telomeric
ends of chromosomes with maximum association in the S phase
8-10 nt binding mode
13-22 nt binding mode
30 nt binding mode
Figure 4. Schematic models of the RPA binding to ssDNA containing
4-SdT substitution in the indicated positions under different binding
modes. In the 30 nt binding mode complex, small RPA subunit p14
(DBD E) can be positioned near the 30 -side of ssDNA and contact DNA
directly.

and to play an essential role in telomere maintenance (69). Fur-
ther, it was shown under near-physiological in vitro conditions
that human RPA is able to bind and unfold G-quadruplex struc-
tures formed from a 21 nt human telomeric sequence (48). Anal-
yses by native gel electrophoresis, crosslinking and fluorescence
resonance energy transfer indicated the formation of both 1:1
and 2:1 complexes in which G-quadruplexes are unfolded. In
addition, quadruplex opening by RPA is much faster than
observed with the complementary DNA, demonstrating that this
protein efficiently unfolds G-quartets.
A two-step mechanism accounting for the binding of RPA to
G-quadruplexes was proposed (48). In this model, RPA initially
takes advantage of the natural G4 breathing that exposes ssDNA
at its extremities. RPA then breaks Hoogsteen hydrogen bonds
that stabilize the guanine quartets. Destabilization of a region of
the G4 structure makes possible the binding of a second mole-
cule of RPA. It was also demonstrated that unfolding of telom-
eric G4 by RPA is strongly impaired by small G4-stabilizing
ligands (70). In addition, photocrosslinking experiments eluci-
dated the polar positioning of the RPA protein subunits along
the unfolded G4. RPA1 and RPA2, but not RPA3, interact with
the telomeric G4 and they are arranged from 50 toward 30 . RPA
can bind on each side of the G4 but it unwinds the G4 only from
5’ toward 3’, with the same directionality as for duplex unfold-
ing (71). The 5’ to 3’ unfolding directionality may be explained
in terms of the 5’ to 3’ orientation of RPA subunits along sin-
gle-stranded DNA. Furthermore, RPA was shown to prevent the
formation of G-quadruplex structures at lagging-strand telomeres
to promote shelterin association and facilitate telomerase action
Figure 5. XPA and RPA location on the open DNA duplex provides
correct topography of the NER preincision complex. RPA bound to
undamaged strand and XPA located on the 5'-side from a lesion near to
ss/ds junction in the damaged bubble recruit the XPG and XPF-ERCC1
endonucleases, respectively, to proper positions to perform dual incision
of the damaged DNA strand.
Figure 6. Proposed location of the RPA and XPA proteins in the pre- and postexcision complexes. After XPF-ERCC1 incision, RPA remains bound
with the undamaged strand in the gap created. RPA can be translocated on the flap when gap is shortened during the resynthesis step.
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Photochemistry and Photobiology, 2020, 96 445
at telomeres (72). These and other data (73) point to the involve-
ment of RPA in regulation of telomere maintenance.
RPA POSITIONING ON THE NER DNA
INTERMEDIATES
RPA is one of the inherent participants of the NER process
which is responsible for removing from DNA UV-induced
pyrimidine dimers and a wide range of the chemically diverse
bulky lesions distorting DNA duplex. Repair process follows
through sequential stages including damage detection, helix
opening, dual incision of the damaged strand 5’ and 3’ to the
lesion, release of the 24-32 nt oligonucleotide, gap-filling DNA
synthesis and ligation. In this process, RPA plays an integral role
in damage recognition preceding the dual incision of a damaged
strand, and then again in postexcision repair synthesis
(20,74,75). Moreover, RPA, along with the TFIIH, was found to
be bound with excised damaged DNA fragments (76).
One of the earliest applications of photoaffinity labeling to
study RPA interaction with the NER intermediates was the
experiments using model 24 bp oligonucleotides containing a
single 1,3-d(GTG)-cisplatin modification and 5-iodo-2’-deoxyuri-
dine (5-IdU) as a crosslinking reagent (77). The 70 kDa subunit
of RPA was crosslinked with high efficiency to cisplatin-modi-
fied DNA probes carrying 5-IdU. Crosslinking efficiency
depended on the presence of the DNA lesion and 5-IdU posi-
tions. Examination of the crosslinking efficiency in dependence
on the position of the 5-IdU revealed that the major contact
points for p70 located to the 5’-side of the DNA lesion on the
damaged strand. The interaction of XPA, the main RPA partner
in the NER process, with the same DNA structures was also ana-
lyzed. Under the conditions tested XPA alone formed a protein–
DNA crosslink only with low efficiency, exhibiting no prefer-
ence for any 5-IdU-position on the DNA substrate. In samples
containing both RPA and XPA, it was only RPA that underwent
significant crosslinking. The presence of XPA affected neither
the specificity nor the crosslinking yield compared with RPA
alone.
Further, this approach was extended and applied to analyze
RPA and XPA interactions with DNA intermediates during dif-
ferent stages of the NER process. A set of 48-mer oligonu-
cleotides bearing fluorescein residue as the lesion and/or 5I-dU
at a definite position as the photoreactive group was used to cre-
ate DNA duplex and 15 nt bubble DNA which mimic intermedi-
ates of the initial and preincision stages of the NER process,

446 Nadejda I. Rechkunova and Olga I. Lavrik
respectively (78,79). The size of bubble equal to 15 nt is in
agreement with the size of bubble opened by TFIIH in the dam-
aged DNA duplex (80). Crosslinking experiments with both
DNA duplex and bubbled DNA display higher yields of DNA-
protein adducts of RPA and XPA with undamaged strand. The
data on DNA duplex modification disagree with previous results
(77) claiming RPA and XPA crosslinks mainly to 5I-dUMP resi-
dues in the damaged strand. This difference is likely due to the
use of different lengths and/or sequences of DNA duplexes and/
or different types of lesions. Each can influence DNA secondary
structure, resulting in changes of the DNA-protein binding sur-
face. It should be noted that protein crosslinking to the cisplatin-
damaged DNA could occur via direct crosslinking to cisplatin
which was shown to act as an effective photoinduced crosslink-
ing agent (81) and could increase the level of crosslinking to
damaged strand. RPA crosslinking to undamaged strand was also
demonstrated using DNA duplex with cholesterol lesion (82).
The model of RPA and XPA positioning on DNA within the
preincision NER complex was proposed based on crosslinks
combined with the EMSA and nuclease footprinting experiments
(Fig. 5). It was assumed (79) that precise positioning RPA bound
to undamaged strand and XPA located on the 5’-side from a
lesion near to ss/ds junction in the damaged bubble can provide
proper orientation of the XPF-ERCC1 and XPG endonucleases
which perform dual incision of the damaged DNA strand.
From the NER factors, only RPA participates during all stages
of the repair process. Using 60-mer DNA duplexes containing a 31
nt flap with the fluorescein residue mimicking the bulky lesion and
a 26 or 10 nt gap, RPA and XPA interactions with intermediates of
the NER late stages were analyzed (83). The structure containing a
damaged flap and a 26 nt gap imitates the DNA intermediate aris-
ing in the NER process after damaged strand cleavage by XPF-
ERCC1; DNA with the same flap and a 10 nt gap can be attributed
to the intermediate of the subsequent partial gap filling (84).
Crosslinking with 5-I-dUMP containing flap-gap26 DNA revealed
that RPA interacts with the ssDNA track in the gap and does not
utilize the flap (83). Based on this study, it may be supposed that,
after XPF-ERCC1 incision, RPA remains bound with the undam-
aged strand in the gap created and can be translocated on the flap
when gap is shortened during the resynthesis step (Fig. 6). It
should be noted that RPA was shown to bind effectively long flap
within the DNA structure lacking a gap (85).
CONCLUSIONS
Photochemical reactions play an essential role in cellular metabo-
lism. UV irradiation damages genomic DNA and can also induce
crosslinks within DNA-protein complexes. An ability of nucleic
acids to form covalent adducts with amino acid residues located
in close proximity was utilized in photoaffinity labeling method
for investigation of protein–nucleic acids interactions. This
approach was developing during several decades with both natu-
ral chromophores (e.g., 4-thiothymine incorporated into the DNA
or nucleic bases themselves) and chemically synthesized nucleo-
tide analogues. Identification of the formed covalent bonds can
help to define the interface between the DNA and the protein,
providing a better understanding of the interactions that occur in
the DNA-protein complex. For more than 40 years, Dr. Alain
Favre contributed to the development of photoreactive nucleotide
derivatives and their application for the study of protein–nucleic
acids interactions.
17511097, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/php.13222 by Cochrane Colombia, Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
This review summarizes data on the application of photoreac-
tive DNA probes as a tool to analyze the interaction of RPA
with the DNA structures mimicking intermediates of replication
and repair processes. The obtained data were important to pro-
pose molecular mechanisms of protein–nucleic acid interactions
in DNA replication, repair and telomere maintenance. Crosslink-
ing technique combined with mass-spectrometry analysis as well
as with structural data seems to be very promising approach for
the discovery of new proteins interacting with specific DNA
structures in cellular extracts of different sources and for deeper
understanding of the architecture of multiprotein complexes oper-
ating in cellular DNA metabolism.
Acknowledgments—The authors are thankful to Dr. Jean Cadet for the
invitation to publish this review and to Dr. Yuliya Krasikova for the
assistant in the manuscript preparation. The work was supported by the
Russian Science Foundation (project #19-14-00107).
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AUTHOR BIOGRAPHIES
Nadejda I. Rechkunova
obtained her Ph.D. degree
in biochemistry at
Novosibirsk Institute of
Bioorganic Chemistry
(now Institute of Chemi-
cal Biology and Funda-
mental Medicine of the
Siberian Branch of the
Russian Academy of Sci-
ences, ICBFM SB RAS).
Her postdoctoral studies
were devoted to develop-
ment of new approaches
for the selective labeling
of DNA polymerases and
replication protein A by photoreactive DNA intermediates of DNA repli-
cation and repair. Now, she is a senior researcher in the Laboratory of
Bioorganic Chemistry of Enzymes at ICBFM SB RAS. Current research
activities of Dr. Rechkunova focus on the compositions and DNA–pro-
tein and protein–protein interactions in nucleotide excision repair com-
plexes and their regulation by poly(ADP-ribosyl)ation.
17511097, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/php.13222 by Cochrane Colombia, Wiley Online Library on [12/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Photochemistry and Photobiology, 2020, 96 449
Olga I. Lavrik obtained
her Ph.D. degree in bioor-
ganic chemistry at the
Institute of Bioorganic
Chemistry of the Russian
Academy of Sciences
(Moscow). She is head of
the Laboratory of Bioor-
ganic Chemistry of
Enzymes at ICBFM SB
RAS, professor in the
Department of Molecular
Biology at the Novosi-
birsk State University and
Corresponding Member of
the Russian Academy of
Sciences. Her main
research interests focus on the DNA repair mechanisms and their contri-
butions to human health and longevity. She is author/co-author of 450
peer-reviewed articles and book chapters. Several times during 1990-
2003 years, she was invited as visiting professor of the University of
Pierre and Marie Curie in Paris where she was working with Prof. Alain
Favre in his laboratory at the Institute Jacques Monod on the study of
protein–nucleic acids interactions using photoreactive tRNA and DNA.
This collaboration resulted in more than 20 joint publications.