Structural basis for the recognition of DNA repair proteins UNG2, XPA, and RAD52 by replication factor RPA

Replication protein A (RPA), the nuclear ssDNA-binding protein in eukaryotes, is essential to DNA replication, recombination, and repair. We have shown that a globular domain at the C terminus of subunit RPA32 contains a specific surface that interacts in a similar manner with the DNA repair enzyme UNG2 and repair factors XPA and RAD52, each of which functions in a different repair pathway. NMR structures of the RPA32 domain, free and in complex with the minimal interaction domain of UNG2, were determined, defining a common structural basis for linking RPA to the nucleotide excision, base excision, and recombinational pathways of repairing damaged DNA. Our findings support a hand-off model for the assembly and coordination of different components of the DNA repair machinery.     

Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between RPA32, RPA70, Rad52 and DNA

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential for DNA replication, and plays important roles in DNA repair and DNA recombination. Rad52 and RPA, along with other members of the Rad52 epistasis group of genes, repair double-stranded DNA breaks (DSBs). Two repair pathways involve RPA and Rad52, homologous recombination and single-strand annealing. Two binding sites for Rad52 have been identified on RPA. They include the previously identified C-terminal domain (CTD) of RPA32 (residues 224-271) and the newly identified domain containing residues 169-326 of RPA70. A region on Rad52, which includes residues 218-303, binds RPA70 as well as RPA32. The N-terminal region of RPA32 does not appear to play a role in the formation of the RPA:Rad52 complex. It appears that the RPA32CTD can substitute for RPA70 in binding Rad52. Sequence homology between RPA32 and RPA70 was used to identify a putative Rad52-binding site on RPA70 that is located near DNA-binding domains A and B. Rad52 binding to RPA increases ssDNA affinity significantly. Mutations in DBD-D on RPA32 show that this domain is primarily responsible for the ssDNA binding enhancement. RPA binding to Rad52 inhibits the higher-order self-association of Rad52 rings. Implications for these results for the "hand-off" mechanism between protein-protein partners, including Rad51, in homologous recombination and single-strand annealing are discussed.     

Nucleotide excision repair by mutant xeroderma pigmentosum group A (XPA) proteins with deficiency in interaction with RPA

The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays, an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an N-terminal deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion. Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.     

Analysis of uracil DNA glycosylase (UNG2) stimulation by replication protein A (RPA) at ssDNA-dsDNA junctions

Replication Protein A (RPA) is a single-stranded DNA binding protein that interacts with DNA repair proteins including Uracil DNA Glycosylase (UNG2). Here, I report DNA binding and activity assays using purified recombinant RPA and UNG2. Using synthetic DNA substrates, RPA was found to promote UNG2's interaction with ssDNA-dsDNA junctions regardless of the DNA strand polarity surrounding the junction. RPA stimulated UNG2's removal of uracil bases paired with adenine or guanine in DNA as much as 17-fold when the uracil was positioned 21 bps from ssDNA-dsDNA junctions, and the largest degree of UNG2 stimulation occurred when RPA was in molar excess compared to DNA. I found that RPA becomes sequestered on ssDNA regions surrounding junctions which promotes its spatial targeting of UNG2 near the junction. However, when RPA concentration exceeds free ssDNA, RPA promotes UNG2's activity without spatial constraints in dsDNA regions. These effects of RPA on UNG2 were found to be mediated primarily by interactions between RPA's winged-helix domain and UNG2's N-terminal domain, but when the winged-helix domain is unavailable, a secondary interaction between UNG2's N-terminal domain and RPA can occur. This work supports a widespread role for RPA in stimulating uracil base excision repair.     

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.     

Interaction of nucleotide excision repair factors XPC-HR23B, XPA, and RPA with damaged DNA

The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.     

Functional studies on the interaction between human replication protein A and Xeroderma pigmentosum group A complementing protein (XPA).

The human replication protein A (RPA; also known as human single-stranded DNA binding protein, HSSB) is a multisubunit complex (70, 34 and 11 kDa subunits) involved in the three processes of DNA metabolism; replication, repair, recombination. We found that both 34 and 70 kDa subunits (p34 and p70, respectively), of RPA interacts with the Xeroderma pigmentosum group A complementing protein (XPA), a protein that specifically recognizes UV-damaged DNA. Our mutational analysis indicated that no particular domains of RPA p70 were essential for its interaction with XPA. We also examined the effect of this XPA-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the crude extract and monopolymerase system. XPA inhibited SV40 DNA replication in vitro through its interaction with RPA. Taken together, these results suggest that there is a role for RPA in the regulation of DNA metabolism through its ability to modulate the interactions of proteins involved in the processes of DNA metabolism.     

Molecular anatomy of the human excision nuclease assembled at sites of DNA damage

Human nucleotide excision repair is initiated by six repair factors (XPA, RPA, XPC-HR23B, TFIIH, XPF-ERCC1, and XPG) which sequentially assemble at sites of DNA damage and effect excision of damage-containing oligonucleotides. We here describe the molecular anatomy of the human excision nuclease assembled at the site of a psoralen-adducted thymine. Three polypeptides, primarily positioned 5' to the damage, are in close physical proximity to the psoralen lesion and thus are cross-linked to the damaged DNA: these proteins are RPA70, RPA32, and the XPD subunit of TFIIH. While both XPA and XPC bind damaged DNA and are required for XPD cross-linking to the psoralen-adducted base, neither XPA nor XPC is cross-linked to the psoralen adduct. The presence of other repair factors, in particular TFIIH, alters the mode of RPA binding and the position of its subunits relative to the psoralen lesion. Based on these results, we propose that RPA70 makes the initial contact with psoralen-damaged DNA but that within preincision complexes, it is RPA32 and XPD that are in close contact with the lesion.     

Sequential binding of DNA repair proteins RPA and ERCC1 to XPA in vitro.

Recent studies have shown that many proteins are involved in the early steps of nucleotide excision repair and that there are some interactions between nucleotide excision repair proteins, suggesting that these interactions are important in the reaction mechanism. The xeroderma pigmentosum group A protein (XPA) was shown to bind to the replication protein A (RPA) or the excision repair cross complementing rodent repair deficiency group 1 protein (ERCC1), and these interactions might be involved in the damage-recognition and/or incision steps, of nucleotide excision repair. Here we show that the XPA regions required for the binding to the 70 and 34 kDa subunits of RPA are located in the middle and on N-terminal regions of XPA, respectively. These regions do not overlap with the ERCC1-binding region of XPA, and a ternary protein complex of RPA, XPA and ERCC1 was detected in vitro. In addition, using the surface plasmon resonance biosensor, the binding of RPA and ERCC1 to XPA was investigated. The dissociation constants (KD) of RPA and ERCC1 with XPA were 1.9 x 10(-8 )and 2.5 x 10(-7) M, respectively. Moreover, our results suggest the sequential binding of RPA and ERCC1 to XPA.     

Xeroderma pigmentosum complementation group A protein (XPA) modulates RPA-DNA interactions via enhanced complex stability and inhibition of strand separation activity

Replication protein A (RPA) participates in many cellular functions including DNA replication and nucleotide excision repair. A direct interaction between RPA and the xeroderma pigmentosum group A protein (XPA) facilitates the assembly of a preincision complex during the processing of DNA damage by the nucleotide excision repair pathway. We demonstrate here the formation of a ternary RPA, XPA, and duplex cisplatin-damaged DNA complex as is evident by electrophoretic supershift analysis. The RPA-XPA complex displays modest specificity for damaged versus undamaged duplex DNA, and the RPA-XPA complex displays a greater affinity for binding duplex cisplatin-damaged DNA when compared with the RPA or XPA proteins alone, consistent with previous results. Using DNA denaturation assays, we demonstrate that the role of XPA is in the stabilization of the duplex DNA structure via inhibition of the strand separation activity of RPA. Rapid kinetic analysis indicates that the bimolecular k(on) of the RPA-XPA complex is 2.5-fold faster than RPA alone for binding a duplex cisplatin-damaged DNA. The dissociation rate, k(off), of the RPA-XPA complex is slower than that of the RPA protein alone, suggesting that the XPA protein stabilizes the initial binding of RPA to duplex DNA as well as maintaining the integrity of the duplex DNA. Interestingly, XPA has no effect on the k(on) of RPA for a single-stranded 40-mer DNA.     

An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.

Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential.     

RPA stabilizes the XPA-damaged DNA complex through protein-protein interaction

The xeroderma pigmentosum group A complementing protein (XPA) and eukaryotic replication protein A (RPA) are among the major damage-recognition proteins involved in the early stage of nucleotide excision repair (NER). XPA and RPA are able to bind damaged DNA independently, although RPA interaction stimulates XPA binding to damaged DNA [Li, L., Lu, X., Peterson, C. A., and Legerski, R. J. (1995) Mol. Cell. Biol. 15, 5396-5402 (1); Stigger, E., Drissi, R., and Lee, S.-H. (1998) J. Biol. Chem. 273, 9337-9343 (2)]. In this study, we used surface plasmon resonance (SPR) analysis to investigate the interaction of XPA and RPA with two major types of UV-damaged DNA: the (6-4) photoproduct and the cis-syn cyclobutane dimer of thymidine. Both XPA and RPA preferentially bind to (6-4) photoproduct-containing duplex DNA over cis-syn cyclobutane dimer-containing DNA. The binding of XPA to (6-4) photoproduct was weak (K(D) = 2.13 x 10(-)(8) M), whereas RPA showed a very stable interaction with (6-4) photoproduct (K(D) = 2. 02 x 10(-)(10) M). When XPA and RPA were incubated together, the stability of the XPA-damaged DNA interaction was significantly enhanced by wild-type RPA. On the other hand, mutant RPA (RPA:p34Delta33C) defective in its interaction with XPA failed to stabilize XPA-damaged DNA complex. Taken together, our results suggest that a role for RPA in UV-damage recognition is to stabilize XPA-damaged DNA complex through protein-protein interaction.     

The UNG2 Arg88Cys variant abrogates RPA-mediated recruitment of UNG2 to single-stranded DNA

In human cell nuclei, UNG2 is the major uracil-DNA glycosylase initiating DNA base excision repair of uracil. In activated B cells it has an additional role in facilitating mutagenic processing of AID-induced uracil at Ig loci and UNG-deficient patients develop hyper-IgM syndrome characterized by impaired class-switch recombination and disturbed somatic hypermutation. How UNG2 is recruited to either error-free or mutagenic uracil processing remains obscure, but likely involves regulated interactions with other proteins. The UNG2 N-terminal domain contains binding motifs for both proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), but the relative contribution of these interactions to genomic uracil processing is not understood. Interestingly, a heterozygous germline single-nucleotide variant leading to Arg88Cys (R88C) substitution in the RPA-interaction motif of UNG2 has been observed in humans, but with unknown functional relevance. Here we demonstrate that UNG2-R88C protein is expressed from the variant allele in a lymphoblastoid cell line derived from a heterozygous germ line carrier. Enzyme activity as well as localization in replication foci of UNG2-R88C was similar to that of WT. However, binding to RPA was essentially abolished by the R88C substitution, whereas binding to PCNA was unaffected. Moreover, we show that disruption of the PCNA-binding motif impaired recruitment of UNG2 to S-phase replication foci, demonstrating that PCNA is a major factor for recruitment of UNG2 to unperturbed replication forks. Conversely, in cells treated with hydroxyurea, RPA mediated recruitment of UNG2 to stalled replication forks independently of functional PCNA binding. Modulation of PCNA- versus RPA-binding may thus constitute a functional switch for UNG2 in cells subsequent to genotoxic stress and potentially also during the processing of uracil at the immunoglobulin locus in antigen-stimulated B cells.     

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.     

Post-replicative base excision repair in replication foci.

Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.     

Polarity of human replication protein A binding to DNA

Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3′-end of the upstream oligonucleotide or at the 5′-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5′-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3′-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer–template systems containing 5′- or 3′-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.     

Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair

The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA–protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5′-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5′-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5′-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG.     

Order of assembly of human DNA repair excision nuclease.

Human excision nuclease removes DNA damage by concerted dual incisions bracketing the lesion. The dual incisions are accomplished by sequential and partly overlapping actions of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF.ERCC1. Of these, RPA, XPA, and XPC have specific binding affinity for damaged DNA. To learn about the role of these three proteins in damage recognition and the order of assembly of the excision nuclease, we measured the binding affinities of XPA, RPA, and XPC to a DNA fragment containing a single (6-4) photoproduct and determined the rate of damage excision under a variety of reaction conditions. We found that XPC has the highest affinity to DNA and that RPA has the highest selectivity for damaged DNA. Under experimental conditions conducive to binding of either XPA + RPA or XPC to damaged DNA, the rate of damage removal was about 5-fold faster for reactions in which XPA + RPA was the first damage recognition factor presented to DNA compared with reactions in which XPC was the first protein that had the opportunity to bind to DNA. We conclude that RPA and XPA are the initial damage sensing factors of human excision nuclease.     

NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair

In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA–XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER.