Pavel E. Pestryakov

Coordinated Regulation of Replication Protein A Activities by Its Subunits p14 and p32

The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPAΔp14, consisting of p70 and p32. RPAΔp14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPAΔp14 complex p32 only minimally recognized the 3′-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPAΔp14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase α-primase, it did not support cell-free SV40 DNA replication. This inability manifested itself in a failure to support both the primer synthesis and primer elongation reactions. These data reveal that efficient binding and correct positioning of the RPA complex on single-stranded DNA requires all three subunits to support DNA replication.

Comparative Analysis of Interaction of Human and Yeast DNA Damage Recognition Complexes with Damaged DNA in Nucleotide Excision Repair

Background: XPC-RAD23B and Rad4-Rad23 proteins are primary damage recognition factors in nucleotide excision repair in human and yeast cells, respectively. Results: XPC-RAD23B and Rad4-Rad23 have contacts with damaged DNA in the same positions. Conclusion: Both proteins reveal similar topography in the complex with damaged DNA in solution. Significance: This study fills the gap between biochemical results for XPC-RAD23B and x-ray data for Rad4-Rad23. The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (KD ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nm, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms.

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double‐stranded but also in single‐stranded DNA. Here, we show that proteins participating in DNA damage response, YB‐1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt‐long double‐stranded DNA. Both RPA and YB‐1 inhibited AP site cleavage by NEIL1 when the AP site was located in single‐stranded DNA. Taking into account a direct interaction of YB‐1 with the AP site, located in single‐stranded DNA, and the high affinity of both YB‐1 and RPA for single‐stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1. Copyright

Essential functions of the 32 kDa subunit of yeast replication protein A

Replication protein A (RPA) is a heterotrimeric (70, 32 and 14 kDa subunits), single-stranded DNA-binding protein required for cellular DNA metabolism. All subunits of RPA are essential for life, but the specific functions of the 32 and 14 kDa subunits remains unknown. The 32 kDa subunit (RPA2) has multiple domains, but only the central DNA-binding domain (called DBD D) is essential for life in Saccharomyces cerevisiae. To define the essential function(s) of RPA2 in S. cerevisiae, a series of site-directed mutant forms of DBD D were generated. These mutant constructs were then characterized in vitro and in vivo. The mutations had minimal effects on the overall structure and activity of the RPA complex. However, several mutants were shown to disrupt crosslinking of RPA2 to DNA and to dramatically lower the DNA-binding affinity of a RPA2-containing subcomplex. When introduced into S. cerevisiae, all DBD D mutants were viable and supported normal growth rates and DNA replication. These findings indicate that RPA2–DNA interactions are not essential for viability and growth in S. cerevisiae. We conclude that DNA-binding activity of RPA2 is dispensable in yeast and that the essential function of DBD D is intra- and/or inter-protein interactions.

Y-box-binding protein 1 as a non-canonical factor of base excision repair.

Base excision repair (BER) is a flagship DNA repair system responsible for maintaining genome integrity. Apart from basal enzymes, this system involves several accessory factors essential for coordination and regulation of DNA processing during substrate channeling. Y-box-binding protein 1 (YB-1), a multifunctional factor that can interact with DNA, RNA, poly(ADP-ribose) and plenty of proteins including DNA repair enzymes, is increasingly considered as a non-canonical protein of BER. Here we provide quantitative characterization of YB-1 physical interactions with key BER factors such as PARP1, PARP2, APE1, NEIL1 and pol β and comparison of the full-length YB-1 and its C-terminally truncated nuclear form in regard to their binding affinities for BER proteins. Data on functional interactions reveal strong stimulation of PARP1 autopoly(ADP-ribosyl)ation and inhibition of poly(ADP-ribose) degradation by PARG in the presence of YB-1. Moreover, YB-1 is shown to stimulate AP lyase activity of NEIL1 and to inhibit dRP lyase activity of pol β on model DNA duplex structure. We also demonstrate for the first time YB-1 poly(ADP-ribosyl)ation in the presence of RNA.

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

Long linear DNA analogs of nucleotide excision repair (NER) substrates have been synthesized. They are 137-mer duplexes containing in their internal positions nucleotides with bulky substitutes imitating lesions with fluorochloroazidopyridyl and fluorescein groups introduced using spacer fragments at the 4N and 5C positions of dCMP and dUMP (Fap-dC- and Flu-dU-DNA) and DNA containing a (+)-cis-stereoisomer of benzo[a]pyrene-N2-deoxyguanidine (BP-dG-DNA, 131 bp). The interaction of the modified DNA duplexes with the proteins of NER-competent HeLa extract was investigated. The substrate properties of the model DNA in the reaction of specific excision were shown to vary in the series Fap-dC-DNA << Flu-dU-DNA < BP-dG-DNA. During the experiments on affinity modification of the proteins of NER-competent extract, Fap-dC-DNA (137 bp) containing a 32P-label in the photoactive nucleotide demonstrated properties of a highly efficient and selective probe. The set of the main targets of labeling included polypeptides of the extract with the same values of apparent molecular weights (35–90 kDa) as when using the shorter (48 bp) Fap-dC-DNA. Besides, some of the extract proteins were shown capable of specific and effective interaction with the long analog of NER substrate. Electrophoretic mobility of these proteins coincided with the mobilities of DNA-binding subunits of XPC-HR23B and PARP1 (∼127 and T]115 kDa, respectively). The 115-kDa target protein was identified as PARP1 using NAD+-based functional testing. The results suggest that the linear Fap-dC-DNA is an unrepairable substrate analog that can compete with effective NER substrates in the binding of the proteins responsible for lesion recognition and excision.

Human and yeast DNA damage recognition complexes bind with high affinity DNA structures mimicking in size transcription bubble

The human XPC‐RAD23B complex and its yeast ortholog, Rad4‐Rad23, are the primary initiators of global genome nucleotide excision repair. In this study, two types of DNA binding assays were used for the detailed analysis of interaction of these proteins with damaged DNA. An electrophoretic mobility shift assay revealed that human and yeast orthologs behave similarly in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed using fluorescent depolarization measurements. The XPC‐RAD23B and the Rad4‐Rad23 proteins bind to the damaged 15 nt bubble‐DNA structure mimicking in size the “transcription bubble” DNA intermediate with the highest affinity (KD values ~10‐10 M or less) that is reduced in the following order: damaged bubble > undamaged bubble > damaged duplex > undamaged duplex. The affinity of XPC/Rad4 for various DNAs was shown to correlate with DNA bending angle. The results obtained show clearly that more deviation from regular DNA structure leads to higher XPC/Rad4 affinity. Copyright

Interaction of nucleotide excision repair protein XPC—RAD23B with DNA containing benzo[a]pyrene-derived adduct and apurinic/apyrimidinic site within a cluster

The combined action of reactive metabolites of benzo[a]pyrene (B[a]P) and oxidative stress can lead to cluster-type DNA damage that includes both a bulky lesion and an apurinic/apyrimidinic (AP) site, which are repaired by the nucleotide and base excision repair mechanisms — NER and BER, respectively. Interaction of NER protein XPC—RAD23B providing primary damage recognition with DNA duplexes containing a B[a]P-derived residue linked to the exocyclic amino group of a guanine (BPDE-N2-dG) in the central position of one strand and AP site in different positions of the other strand was analyzed. It was found that XPC—RAD23B crosslinks to DNA containing (+)-trans-BPDE-N2-dG more effectively than to DNA containing cis-isomer, independently of the AP site position in the opposite strand; protein affinity to DNA containing one of the BPDE-N2-dG isomers depends on the AP site position in the opposite strand. The influence of XPC—RAD23B on hydrolysis of an AP site clustered with BPDE-N2-dG catalyzed by the apurinic/apyrimidinic endonuclease 1 (APE1) was examined. XPC—RAD23B was shown to stimulate the endonuclease and inhibit the 3′–5′ exonuclease activity of APE1. These data demonstrate the possibility of cooperation of two proteins belonging to different DNA repair systems in the repair of cluster-type DNA damage.

Inhibition of abasic site cleavage in bubble DNA by multifunctional protein YB-1

Y‐box binding protein 1 (YB‐1) is widely known to participate in a multiple DNA and RNA processing events in the living cell. YB‐1 is also regarded as a putative component of DNA repair. This possibility is supported by relocalization of YB‐1 into the nucleus following genotoxic stress. Increased affinity of YB‐1 for damaged DNA, especially in its single‐stranded form, and its functional interaction with proteins responsible for the initiation of apurinic/apyrimidinic (AP) site repair, namely, AP endonuclease 1 and DNA glycosylase NEIL1, suggest that YB‐1 could be involved in the repair of AP sites as a regulatory protein. Here we show that YB‐1 has a significant inhibitory effect on the cleavage of AP sites located in single‐stranded DNA and in DNA bubble structures. Such interference may be considered as a possible mechanism to prevent single‐stranded intermediates of DNA replication, transcription and repair from being converted into highly genotoxic DNA strand breaks, thus allowing the cell to coordinate different DNA processing mechanisms. Copyright

Interaction of nucleotide excision repair proteins with DNA containing bulky lesion and apurinic/apyrimidinic site

The interaction of nucleotide excision repair (NER) proteins (XPC-HR23b, RPA, and XPA) with 48-mer DNA duplexes containing the bulky lesion-mimicking fluorescein-substituted derivative of dUMP (5-{3-[6-(carboxyamidofluo-resceinyl)amidocapromoyl]allyl}-2′-deoxyuridine-5′-monophosphate) in a cluster with a lesion of another type (apurinic/apyrimidinic (AP) site) has been studied. It is shown that XPC-HR23b is modified to a greater extent by the DNA duplex containing an AP site opposite nucleotide adjacent to the fluorescein residue than by DNA containing an AP site shifted to the 3′-or 5′-end of the DNA strand. The efficiency of XPA modification by DNA duplexes containing both AP site and fluorescein residue is higher than that by DNA lacking the bulky lesion; the modification pattern in this case depends on the AP site position. In accordance with its major function, RPA interacts more efficiently with single-stranded DNA than with DNA duplexes, including those bearing bulky lesions. The observed interaction between the proteins involved in nucleotide excision repair and DNA structures containing a bulky lesion processed by NER and the AP site repaired via base excision repair may be significant for both these repair pathways in cells and requires the specific sequence of repair of clustered DNA lesions.