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 ß 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 ß on model DNA duplex structure. We also demonstrate for the first time YB-1 poly(ADP-ribosyl)ation in the presence of RNA.

Poly(ADP-ribosyl)ation as a new posttranslational modification of YB-1.

Multifunctional Y-box binding protein 1 (YB-1) is actively studied as one of the components of cellular response to genotoxic stress. However, the precise role of YB-1 in the process of DNA repair is still obscure. In the present work we report for the first time new posttranslational modification of YB-1 - poly(ADP-ribosyl)ation, catalyzed by one of the main regulatory enzymes of DNA repair - poly(ADP-ribose)polymerase 1 (PARP1) in the presence of model DNA substrate carrying multiple DNA lesions. Therefore, poly(ADP-ribosyl)ation of YB-1 catalyzed with PARP1, can be stimulated by damaged DNA. The observed property of YB-1 underlines its ability to participate in the DNA repair by its involvement in the regulatory cascades of DNA repair.

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.

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.

Comparative analysis of interaction of human and yeast DNA damage recognition complexes with damaged DNA in nucleotide excision repair.

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 (K(D) ∼ (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.

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.

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, RPADeltap14, consisting of p70 and p32. RPADeltap14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPADeltap14 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 RPADeltap14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase alpha-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.

Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains.

The human nuclear single-stranded (ss) DNA- binding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein-DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5'-protruding ssDNA tails of 10, 20 and 30 nt. The crosslinks were generated using either a 'zero-length' photoreactive group (4-thio-2'-deoxyuridine-5'-monophosphate) embedded in the 3' end of the DNA primer, or a group connected to the 3' end by a lengthy linker (5-[N-[N-(4-azido-2,5-difluoro-3- chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-monophosphate). In the absence of two major DBDs, p70A and p70B, the RPA trimerization core (p70C.p32D.p14) was capable of correctly recognizing the primer- template junction and adopting an orientation similar to that in native RPA. Both p70C and p32D contributed to this recognition. However, the domain contribution differed depending on the size of the ssDNA. In contrast with the trimerization core, the RPA dimerization core (p32D.p14) was incapable of detectably recognizing the DNA- junction structures, suggesting an orchestrating role for p70C in this process.

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.