Interaction and stimulation of human FEN-1 nuclease activities by heterogeneous nuclear ribonucleoprotein A1 in alpha-segment processing during Okazaki fragment maturation

High-fidelity DNA replication depends on both accurate incorporation of nucleotides in the newly synthesized strand and the maturation of Okazaki fragments. In eukaryotic cells, the latter is accomplished by a series of coordinated actions of a set of structure-specific nucleases, which, with the assistance of accessory proteins, recognize branched RNA/DNA configurations. In the current model of Okazaki fragment maturation, displacement of a 27-nucleotide or longer flap is envisioned to attract replication protein A (RPA), which inhibits flap endonuclease-1 (FEN-1) but stimulates Dna2 nuclease for cleavage. Dna2 cleavage generates a short flap of 5-7 nucleotides, which resists binding by RPA and further cleavage by Dna2. FEN-1 then removes the remaining flap to produce a suitable substrate for ligation. However, FEN-1 is not efficient in cleaving the short flap, and we therefore set out to identify cellular factors that might regulate FEN-1 activity. Through co-immunoprecipitation experiments, we have isolated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which forms a direct complex with FEN-1 and stimulates its enzymatic activities. The stimulation by hnRNP A1 is most dramatic using DNA substrates with short flaps. With longer flap substrates the hnRNP A1 effect is more modest and is suppressed by the addition of RPA. A model is provided to explain the possible in vivo role of this interaction and activity in Okazaki fragment maturation.     

The characterization of a mammalian DNA structure-specific endonuclease.

The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.     

Interaction of replication protein A and flap endonuclease 1 with DNA duplexes containing a nick or flap

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase beta (Pol beta) interaction with DNA. Using Pol beta, a photoreactive dTTP analog was added to the 3' end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5' end of a nick or a flap. Photoaffinity labeling of Pol beta substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol beta-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol beta during long-patch base excision repair.     

Interaction of Replication Protein A with Photoreactive DNA Structures

A new photoreactive oligonucleotide derivative was synthesized with a perfluoroarylazido group attached to the 2'-position of the ribose fragment of the 5'-terminal nucleotide. Using this conjugate, photoreactive DNA duplexes were produced which contained single-stranded regions of different length, single-stranded breaks (nicks), and also ds duplex with a photoreactive group inside one of the chains. These structures imitate DNA intermediates generated at different stages of DNA replication and repair. The interaction of replication protein A (RPA) with the resulting DNA structures was studied using photoaffinity modification and gel retardation assay. Independently of the DNA structure, only the large subunit of RPA (p70) was crosslinked to photoreactive DNAs, and the intensity of its labeling increased with decrease in the size of the single-stranded region and was maximal in the case of the nick-containing DNA structure. By gel retardation, the most effective binding of RPA to this structure was shown, whereas the complexing of RPA with DNA containing the unmodified nick and also with the full duplex containing the photoreactive group inside the chain was significantly less effective. The data suggest that RPA should be sensitive to such damages in the double-stranded DNA structure.     

Biochemical characterization of the WRN-FEN-1 functional interaction

Werner Syndrome is a premature aging disorder characterized by chromosomal instability. Recently we reported a novel interaction of the WRN gene product with human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in pathways of DNA metabolism that are important for genomic stability. To characterize the mechanism for WRN stimulation of FEN-1 cleavage, we have determined the effect of WRN on the kinetic parameters of the FEN-1 cleavage reaction. WRN enhanced the efficiency of FEN-1 cleavage rather than DNA substrate binding. WRN effectively stimulated FEN-1 cleavage on a flap DNA substrate with streptavidin bound to the terminal 3' nucleotide at the end of the upstream duplex, indicating that WRN does not require a free upstream end to stimulate FEN-1 cleavage of the 5' flap substrate. These results indicate that the mechanism whereby WRN stimulates FEN-1 cleavage is distinct from that proposed for the functional interaction between proliferating cell nuclear antigen and FEN-1. To understand the potential importance of the WRN-FEN-1(1) interaction in DNA replication, we have tested the effect of WRN on FEN-1 cleavage of several DNA substrate intermediates that may arise during Okazaki fragment processing. WRN stimulated FEN-1 cleavage of flap substrates with a terminal monoribonucleotide, a long 5' ssDNA tract, and a pseudo-Y structure. The ability of WRN to facilitate FEN-1 cleavage of DNA replication/repair intermediates may be important for the role of WRN in the maintenance of genomic stability.     

Interaction of nucleotide excision repair factors RPA and XPA with DNA containing bulky photoreactive groups imitating damages

Interaction of nucleotide excision repair factors--replication protein A (RPA) and Xeroderma pigmentosum complementing group A protein (XPA)--with DNA structures containing nucleotides with bulky photoreactive groups imitating damaged nucleotides was investigated. Efficiency of photoaffinity modification of two proteins by photoreactive DNAs varied depending on DNA structure and type of photoreactive group. The secondary structure of DNA and, first of all, the presence of extended single-stranded parts plays a key role in recognition by RPA. However, it was shown that RPA efficiently interacts with DNA duplex containing a bulky substituent at the 5 -end of a nick. XPA was shown to prefer the nicked DNA; however, this protein was cross-linked with approximately equal efficiency by single-stranded and double-stranded DNA containing a bulky substituent inside the strand. XPA seems to be sensitive not only to the structure of DNA double helix, but also to a bulky group incorporated into DNA. The mechanism of damage recognition in the process of nucleotide excision repair is discussed.     

Arginine residues 47 and 70 of human flap endonuclease-1 are involved in DNA substrate interactions and cleavage site determination

Flap endonuclease-1 (FEN-1) is a critical enzyme for DNA replication and repair. Intensive studies have been carried out on its structure-specific nuclease activities and biological functions in yeast cells. However, its specific interactions with DNA substrates as an initial step of catalysis are not defined. An understanding of the ability of FEN-1 to recognize and bind a flap DNA substrate is critical for the elucidation of its molecular mechanism and for the explanation of possible pathological consequences resulting from its failure to bind DNA. Using human FEN-1 in this study, we identified two positively charged amino acid residues, Arg-47 and Arg-70 in human FEN-1, as candidates responsible for substrate binding. Mutation of the Arg-70 significantly reduced flap endonuclease activity and eliminated exonuclease activity. Mutation or protonation of Arg-47 shifted cleavage sites with flap substrate and significantly reduced the exonuclease activity. We revealed that these alterations are due to the defects in DNA-protein interactions. Although the effect of the single Arg-47 mutation on binding activities is not as severe as R70A, its double mutation with Asp-181 had a synergistic effect. Furthermore the possible interaction sites of these positively charged residues with DNA substrates were discussed based on FEN-1 cleavage patterns using different substrates. Finally data were provided to indicate that the observed negative effects of a high concentration of Mg(2+) on enzymatic activity are probably due to the competition between the arginine residues and metal ions with DNA substrate since mutants were found to be less tolerant.     

Human replication protein A. The C-terminal RPA70 and the central RPA32 domains are involved in the interactions with the 3'-end of a primer-template DNA

Although the mechanical aspects of the single-stranded DNA (ssDNA) binding activity of human replication protein A (RPA) have been extensively studied, only limited information is available about its interaction with other physiologically relevant DNA structures. RPA interacts with partial DNA duplexes that resemble DNA intermediates found in the processes of DNA replication and DNA repair. Limited proteolysis of RPA showed that RPA associated with ssDNA is less protected against proteases than RPA bound to a partial duplex DNA containing a 5'-protruding tail that had the same length as the ssDNA. Modification of both the 70- and 32-kDa subunits, RPA70 and RPA32, respectively, by photoaffinity labeling indicates that RPA can bind the primer-template junction of partial duplex DNAs by interacting with the 3'-end of the primer. The identification of the protein domains modified by the photoreactive 3'-end of the primer showed 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 these interactions.     

Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA.

In eukaryotic cells, a 5' flap DNA endonuclease activity and a ds DNA 5'-exonuclease activity exist within a single enzyme called FEN-1 [flap endo-nuclease and 5(five)'-exo-nuclease]. This 42 kDa endo-/exonuclease, FEN-1, is highly homologous to human XP-G, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1. These structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap, and its derivative called a pseudo Y-structure. FEN-1 is essential for lagging strand DNA synthesis in Okazaki fragment joining. FEN-1 also appears to be important in mismatch repair. Here we find that human PCNA, the processivity factor for eukaryotic polymerases, physically associates with human FEN-1 and stimulates its endonucleolytic activity at branched DNA structures and its exonucleolytic activity at nick and gap structures. Structural requirements for FEN-1 and PCNA loading provide an interesting picture of this stimulation. PCNA loads on to substrates at double-stranded DNA ends. In contrast, FEN-1 requires a free single-stranded 5' terminus and appears to load by tracking along the single-stranded DNA branch. These physical constraints define the range of DNA replication, recombination and repair processes in which this family of structure-specific nucleases participate. A model explaining the exonucleolytic activity of FEN-1 in terms of its endonucleolytic activity is proposed based on these observations.     

Interaction of Replication Protein A and Flap Endonuclease 1 with DNA Duplexes Containing a Nick or a Flap

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase (Pol) interaction with DNA. Using Pol, a photoreactive dTTP analog was added to the 3" end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5" end of a nick or a flap. Photoaffinity labeling of Pol substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol during long-patch base excision repair.     

Human flap endonuclease-1: conformational change upon binding to the flap DNA substrate and location of the Mg2+ binding site

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is a key enzyme in DNA replication and repair. FEN-1 recognizes the 5'-flap DNA structure and cleaves it, a specialized endonuclease function essential for the processing of Okazaki fragments during DNA replication and for the repair of 5'-end single-stranded tails from nicked double-stranded DNA substrates. Magnesium is a cofactor required for nuclease activity. We have used Fourier transform infrared (FTIR) spectroscopy to better understand how Mg2+ and flap DNA interact with human FEN-1. FTIR spectroscopy provides three fundamentally new insights into the structural changes induced by the interaction of FEN-1 with substrate DNA and Mg2+. First, FTIR difference spectra in the amide I vibrational band (1600-1700 cm(-1)) reveal a change in the secondary structure of FEN-1 induced by substrate DNA binding. Quantitative analysis of the FTIR spectra indicates a 4% increase in helicity upon DNA binding or about 14 residues converted from disordered to helical conformations. The observation that the residues are disordered without DNA strongly implicates the flexible loop region. The conversion to helix also suggests a mechanism for locking the flexible loop region around the bound DNA. This is the first direct experimental evidence for a binding mechanism that involves a secondary structural change of the protein. Second, in contrast with DNA binding, no change is observed in the secondary structure of FEN-1 upon Mg2+ binding to the wild type or to the noncleaving D181A mutant. Third, the FTIR results provide direct evidence (via the carboxylate ligand band at 1535 cm(-1)) that not only is D181 a ligand to Mg2+ in the human enzyme but Mg2+ binding does not occur in the D181A mutant which lacks this ligand.     

Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae

Eukaryotic Okazaki fragments are initiated by a RNA/DNA primer, which is removed before the fragments are joined. Polymerase delta displaces the primer into a flap for processing. Dna2 nuclease/helicase and flap endonuclease 1 (FEN1) are proposed to cleave the flap. The single-stranded DNA-binding protein, replication protein A (RPA), governs cleavage activity. Flap-bound RPA inhibits FEN1. This necessitates cleavage by Dna2, which is stimulated by RPA. FEN1 then cuts the remaining RPA-free flap to create a nick for ligation. Cleavage by Dna2 requires that it enter the 5'-end and track down the flap. Because Dna2 cleaves the RPA-bound flap, we investigated the mechanism by which Dna2 accesses the protein-coated flap for cleavage. Using a nuclease-defective Dna2 mutant, we showed that just binding of Dna2 dissociates the flap-bound RPA. Facile dissociation is specific to substrates with a genuine flap, and will not occur with an RPA-coated single strand. We also compared the cleavage patterns of Dna2 with and without RPA to better define RPA stimulation of Dna2. Stimulation derived from removal of DNA folding in the flap. Apparently, coordinated with its dissociation, RPA relinquishes the flap to Dna2 for tracking in a way that does not allow flap structure to reform. We also found that RPA strand melting activity promotes excessive flap elongation, but it is suppressed by Dna2-promoted RPA dissociation. Overall, results indicate that Dna2 and RPA coordinate their functions for efficient flap cleavage and preparation for FEN1.     

On the roles of Saccharomyces cerevisiae Dna2p and Flap endonuclease 1 in Okazaki fragment processing

Short DNA segments designated Okazaki fragments are intermediates in eukaryotic DNA replication. Each contains an initiator RNA/DNA primer (iRNA/DNA), which is converted into a 5'-flap and then removed prior to fragment joining. In one model for this process, the flap endonuclease 1 (FEN1) removes the iRNA. In the other, the single-stranded binding protein, replication protein A (RPA), coats the flap, inhibits FEN1, but stimulates cleavage by the Dna2p helicase/nuclease. RPA dissociates from the resultant short flap, allowing FEN1 cleavage. To determine the most likely process, we analyzed cleavage of short and long 5'-flaps. FEN1 cleaves 10-nucleotide fixed or equilibrating flaps in an efficient reaction, insensitive to even high levels of RPA or Dna2p. On 30-nucleotide fixed or equilibrating flaps, RPA partially inhibits FEN1. CTG flaps can form foldback structures and were inhibitory to both nucleases, however, addition of a dT(12) to the 5'-end of a CTG flap allowed Dna2p cleavage. The presence of high Dna2p activity, under reaction conditions favoring helicase activity, substantially stimulated FEN1 cleavage of tailed-foldback flaps and also 30-nucleotide unstructured flaps. Our results suggest Dna2p is not used for processing of most flaps. However, Dna2p has a role in a pathway for processing structured flaps, in which it aids FEN1 using both its nuclease and helicase activities.     

Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway

Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) pyrimidine dimer photoproducts in DNA. Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified. Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA glycosylases have also been characterized. Recently, a novel UV damage endonuclease (UVDE) was identified that initiates an excision repair pathway different from previously established repair mechanisms. Homologues of UVDE have been found in eukaryotes as well as in bacteria. In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimidine dimers and (6-4) photoproducts for the characterization of this UV damage repair pathway. After introduction of single-strand breaks at the 5' sides of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins). FEN-1 homologues from humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions. T4 endonuclease V-incised DNA was processed in the same way. Both nicked and flapped DNA substrates with photolesions (the latter may be intermediates in DNA polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1. The data suggest that the two enzymatic activities, UVDE and FEN-1, are part of an alternative excision repair pathway for repair of UV photoproducts.     

Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair

Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular beta sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites.     

Concerted action of exonuclease and Gap-dependent endonuclease activities of FEN-1 contributes to the resolution of triplet repeat sequences (CTG)n- and (GAA)n-derived secondary structures formed during maturation of Okazaki fragments

There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG)n and (GAA)n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG)n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for single-stranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.     

Dna2 is a structure-specific nuclease,with affinity for 5′-flap intermediates

Dna2 is a nuclease/helicase with proposed roles in DNA replication, double-strand break repair and telomere maintenance. For each role Dna2 is proposed to process DNA substrates with a 5′-flap. To date, however, Dna2 has not revealed a preference for binding or cleavage of flaps over single-stranded DNA. Using DNA binding competition assays we found that Dna2 has substrate structure specificity. The nuclease displayed a strong preference for binding substrates with a 5′-flap or some variations of flap structure. Further analysis revealed that Dna2 recognized and bound both the single-stranded flap and portions of the duplex region immediately downstream of the flap. A model is proposed in which Dna2 first binds to a flap base, and then the flap threads through the protein with periodic cleavage, to a terminal flap length of ∼5 nt. This resembles the mechanism of flap endonuclease 1, consistent with cooperation of these two proteins in flap processing.     

DNA polymerase beta -mediated long patch base excision repair. Poly(ADP-ribose)polymerase-1 stimulates strand displacement DNA synthesis.

Recently, photoaffinity labeling experiments with mouse cell extracts suggested that PARP-1 functions as a surveillance protein for a stalled BER intermediate. To further understand the role of PARP-1 in BER, we examined the DNA synthesis and flap excision steps in long patch BER using a reconstituted system containing a 34-base pair BER substrate and five purified human enzymes: uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta, flap endonuclease-1 (FEN-1), and PARP-1. PARP-1 stimulates strand displacement DNA synthesis by DNA polymerase beta in this system; this stimulation is dependent on the presence of FEN-1. PARP-1 and FEN-1, therefore, cooperate to activate long patch BER. The results are discussed in the context of a model for BER sub-pathway choice, illustrating a dual role for PARP-1 as a surveillance protein for a stalled BER intermediate and an activating factor for long patch BER DNA synthesis.     

Use of Modified Flap Structures for Study of Base Excision Repair Proteins

To investigate interactions between proteins participating in the long-patch pathway of base excision repair (BER), DNA duplexes with flap strand containing modifications in sugar phosphate backbone within the flap-forming oligonucleotides were designed. When the flap-forming oligonucleotide consisted of two sequences bridged by a decanediol linker located in the flap strand near the branch point, the efficiency and position of cleavage by flap endonuclease 1 (FEN1) differed from those for natural flap. The cleavage rate of chimeric structure by FEN1 was lower than that of a normal substrate. When we introduced the second modification in the flap-forming oligonucleotide, the cleavage rate decreased significantly. To estimate efficiency of recognition and processing of the chimeric structures by BER proteins, we studied the rate of DNA synthesis by DNA polymerase beta (Pol beta) and the rate of nucleotide excision at the 3'-end of the initiating primer by apurinic/apyrimidinic endonuclease 1 (APE1) compared with those for the natural DNA duplexes. Efficiency of strand-displacement DNA synthesis catalyzed by Pol beta was shown to be higher for flap structures containing non-nucleotide linkers. The chimeric structures were processed by the 3'-exonuclease activity of APE1 with efficiency lower than that for a normal flap structure. Thus, DNA duplexes with modifications in sugar phosphate backbone can be used to mimic intermediates of the long-patch pathway of BER in reconstituted systems containing FEN1. Based on chimeric and natural oligonucleotides, photoreactive DNA structures were designed. The photoreactive dCMP moiety was introduced into the 3'-end of DNA primer via the activity of Pol beta. The photoreactive DNA duplexes--3'-recessed DNA, nicked DNA, and flap structures containing natural and chimeric oligonucleotides--were used for photoaffinity labeling of BER proteins.     

RPA repair recognition of DNA containing pyrimidines bearing bulky adducts

Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has been analyzed using fluorescence spectroscopy and photoaffinity modification. The aim of the present work was to find quantitative characteristics of RPA-ddsDNA interaction and RPA subunits role in this process. The designed DNA structures bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine. J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The reporter group was a fluorescein residue, introduced into the 5'-phosphate end of one duplex-forming DNA strand. RPA-dsDNA association constants and the molar RPA/dsDNA ratio have been calculated based on fluorescence anisotropy measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly depends on the adduct type and varies in the following way: undamaged dsDNA < Antr-dC-ddsDNA < mmdsDNA < FAPdU-, Pyr-dU-ddsDNA < FAP-dC-ddsDNA (K(D) = 68 +/- 1; 25 +/- 6; 13 +/- 1; 8 +/- 2, and 3.5 +/- 0.5 nM correspondingly) but weakly depends on the chain integrity. Interestingly the bulkier lesions not in all cases have a greater effect on RPA affinity to ddsDNA. The experiments on photoaffinity modification demonstrated only p70 of compactly arranged RPA directly interacting with dsDNA. The formation of RPA-ddsDNA covalent adducts was drastically reduced when both strands of DNA duplex contained virtually opposite located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for the effective interaction with dsDNA bearing bulky damages and demonstrates the early NER factors characteristic features underlying strand discrimination capacity and poor activity of the NER system toward double damaged DNA.