Regulatory functions of the N-terminal domain of the 70-kDa subunit of replication protein A (RPA)

Replication protein A (RPA) is the major single-stranded DNA-binding protein in eukaryotes. RPA is composed of three subunits of 70, 32, and 14 kDa. The N-terminal domain of the 70-kDa subunit (RPA70) has weak DNA binding activity, interacts with proteins, and is involved in cellular DNA damage response. To define the mechanism by which this domain regulates RPA function, we analyzed the function of RPA forms containing a deletion of the N terminus of RPA70 and mutations in the phosphorylation domain of RPA (N-terminal 40 amino acids of the 32-kDa subunit). Although each individual mutation has only modest effects on RPA activity, a form combining both phosphorylation mimetic mutations and a deletion of the N-terminal domain of RPA70 was found to have dramatically altered activity. This combined mutant was defective in binding to short single-stranded DNA oligonucleotides and had altered interactions with proteins that bind to the DNA-binding core of RPA70. These results indicate that in the absence of the N-terminal domain of RPA70, a negatively charged phosphorylation domain disrupts the activity of the core DNA-binding domain of RPA. We conclude that the N-terminal domain of RPA70 functions by interacting with the phosphorylation domain of the 32-kDa subunit and blocking undesirable interactions with the core DNA-binding domain of RPA. These studies indicate that RPA conformation is important for regulating RPA-DNA and RPA-protein interactions.     

Replication protein A interactions with DNA. 1. Functions of the DNA-binding and zinc-finger domains of the 70-kDa subunit

Human replication protein A (RPA) is a multiple subunit single-stranded DNA-binding protein that is required for multiple processes in cellular DNA metabolism. This complex, composed of subunits of 70, 32, and 14 kDa, binds to single-stranded DNA (ssDNA) with high affinity and participates in multiple protein-protein interactions. The 70-kDa subunit of RPA is known to be composed of multiple domains: an N-terminal domain that participates in protein interactions, a central DNA-binding domain (composed of two copies of a ssDNA-binding motif), a putative (C-X2-C-X13-C-X2-C) zinc finger, and a C-terminal intersubunit interaction domain. A series of mutant forms of RPA were used to elucidate the roles of these domains in RPA function. The central DNA-binding domain was necessary and sufficient for interactions with ssDNA; however, adjacent sequences, including the zinc-finger domain and part of the N-terminal domain, were needed for optimal ssDNA-binding activity. The role of aromatic residues in RPA-DNA interactions was examined. Mutation of any one of the four aromatic residues shown to interact with ssDNA had minimal effects on RPA activity, indicating that individually these residues are not critical for RPA activity. Mutation of the zinc-finger domain altered the structure of the RPA complex, reduced ssDNA-binding activity, and eliminated activity in DNA replication.     

RPA-like proteins mediate yeast telomere function

Cdc13, Stn1 and Ten1 are essential yeast proteins that both protect chromosome termini from unregulated resection and regulate telomere length. Cdc13, which localizes to telomeres through high-affinity binding to telomeric single-stranded DNA, has been extensively characterized, whereas the contribution(s) of the Cdc13-associated Stn1 and Ten1 proteins to telomere function have remained unclear. We show here that Stn1 and Ten1 are DNA-binding proteins with specificity for telomeric DNA substrates. Furthermore, Stn1 and Ten1 show similarities to Rpa2 and Rpa3, subunits of the heterotrimeric replication protein A (RPA) complex, which is the major single-stranded DNA-binding activity in eukaryotic cells. We propose that Cdc13, Stn1 and Ten1 function as a telomere-specific RPA-like complex. Identification of an RPA-like complex that is targeted to a specific region of the genome suggests that multiple RPA-like complexes have evolved, each making individual contributions to genomic stability.     

The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction

Replication protein A (RPA) is a heterotrimeric (subunits of 70, 32, and 14 kDa) single-stranded DNA-binding protein that is required for DNA replication, recombination, and repair. The 40-residue N-terminal domain of the 32-kDa subunit of RPA (RPA32) becomes phosphorylated during S-phase and after DNA damage. Recently it has been shown that phosphorylation or the addition of negative charges to this N-terminal phosphorylation domain modulates RPA-protein interactions and increases cell sensitivity to DNA damage. We found that addition of multiple negative charges to the N-terminal phosphorylation domain also caused a significant decrease in the ability of a mutant form of RPA to destabilize double-stranded (ds) DNA. Kinetic studies suggested that the addition of negative charges to the N-terminal phosphorylation domain caused defects in both complex formation (nucleation) and subsequent destabilization of dsDNA by RPA. We conclude that the N-terminal phosphorylation domain modulates RPA interactions with dsDNA. Similar changes in DNA interactions were observed with a mutant form of RPA in which the N-terminal domain of the 70-kDa subunit was deleted. This suggested a functional link between the N-terminal domains of the 70- and 32-kDa subunits of RPA. NMR experiments provided evidence for a direct interaction between the N-terminal domain of the 70-kDa subunit and the negatively charged N-terminal phosphorylation domain of RPA32. These findings suggest that phosphorylation causes a conformational change in the RPA complex that regulates RPA function.     

Replication protein A interactions with DNA. 2. Characterization of double-stranded DNA-binding/helix-destabilization activities and the role of the zinc-finger domain in DNA interactions

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellular DNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA), (2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization. The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the melting temperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar, and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions required for ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDa subunit and that other domains of RPA are needed for optimal activity. We conclude that all types of RPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs when RPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPA homologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directly interacts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that a metal ion is required for the function of the zinc-finger motif.     

The 32-kilodalton subunit of replication protein A interacts with menin,the product of the MEN1 tumor suppressor gene

Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.     

Replication protein A interactions with DNA. III. Molecular basis of recognition of damaged DNA

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.     

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.     

The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding.

Replication protein A (RPA), the eukaryote single-stranded DNA-binding protein (SSB), is a heterotrimer. The largest subunit, RPA70, which harbours the major DNA-binding activity, has two DNA-binding domains that each adopt an OB-fold. The complex of the two smaller subunits, RPA32 and RPA14, has weak DNA-binding activity but the mechanism of DNA binding is unknown. We have determined the crystal structure of the proteolytic core of RPA32 and RPA14, which consists of the central two-thirds of RPA32 and the entire RPA14 subunit. The structure revealed that RPA14 and the central part of RPA32 are structural homologues. Each subunit contains a central OB-fold domain, which also resembles the DNA-binding domains in RPA70; an N-terminal extension that interacts with the central OB-fold domain; and a C-terminal helix that mediate heterodimerization via a helix-helix interaction. The OB-fold of RPA32, but not RPA14, possesses additional similarity to the RPA70 DNA-binding domains, supporting a DNA-binding role for RPA32. The discovery of a third and fourth OB-fold in RPA suggests that the quaternary structure of SSBs, which in Bacteria and Archaea are also tetramers of OB-folds, is conserved in evolution. The structure also suggests a mechanism for RPA trimer formation.     

Critical Functions of Rpa3/Ssb3 in S-Phase DNA Damage Responses in Fission Yeast

Replication Protein A (RPA) is a heterotrimeric, single-stranded DNA (ssDNA)–binding complex required for DNA replication and repair, homologous recombination, DNA damage checkpoint signaling, and telomere maintenance. Whilst the larger RPA subunits, Rpa1 and Rpa2, have essential interactions with ssDNA, the molecular functions of the smallest subunit Rpa3 are unknown. Here, we investigate the Rpa3 ortholog Ssb3 in Schizosaccharomyces pombe and find that it is dispensable for cell viability, checkpoint signaling, RPA foci formation, and meiosis. However, increased spontaneous Rad11^Rpa1 and Rad22^Rad52 nuclear foci in ssb3Δ cells indicate genome maintenance defects. Moreover, Ssb3 is required for resistance to genotoxins that disrupt DNA replication. Genetic interaction studies indicate that Ssb3 has a close functional relationship with the Mms1-Mms22 protein complex, which is required for survival after DNA damage in S-phase, and with the mitotic functions of Mus81-Eme1 Holliday junction resolvase that is required for recovery from replication fork collapse. From these studies we propose that Ssb3 plays a critical role in mediating RPA functions that are required for repair or tolerance of DNA lesions in S-phase. Rpa3 orthologs in humans and other species may have a similar function.     

The role for zinc in replication protein A

Heterotrimeric human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA), is a central player in DNA replication, recombination, and repair. The C terminus of the largest subunit, RPA70, contains a putative zinc-binding motif and is implicated in complex formation with two smaller subunits, RPA14 and RPA32. The C-terminal domain of RPA70 (RPA70-CTD) was characterized using proteolysis and x-ray fluorescence emission spectroscopy. The proteolytic core of this domain comprised amino acids 432-616. X-ray fluorescence spectra revealed that RPA70-CTD possesses a coordinated Zn(II). The trimeric complex of RPA70-CTD, the ssDNA-binding domain of RPA32 (amino acids 43-171), and RPA14 had strong DNA binding activity. When properly coordinated with zinc, the trimer's affinity to ssDNA was only 3-10-fold less than that of the ssDNA-binding domain in the middle of RPA70. However, the DNA-binding activity of the trimer was dramatically reduced in the presence of chelating agents. Our data indicate that (i) Zn(II) is essential to stabilize the tertiary structure of RPA70-CTD; (ii) RPA70-CTD possesses DNA-binding activity, which is modulated by Zn(II); and (iii) ssDNA binding by the trimer is a synergistic effect generated by the RPA70-CTD and RPA32.     

Cloning of the large subunit of replication protein A (RPA) from yeast Saccharomyces cerevisiae and its DNA binding activity through redox potential

Eukaryotic replication protein A (RPA) is a single-stranded(ss) DNA binding protein with multiple functions in DNA replication, repair, and genetic recombination. The 70-kDa subunit of eukaryotic RPA contains a conserved four cysteine-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Recently, we described a novel function for the zinc-finger motif in the regulation of human RPA's ssDNA binding activity through reduction-oxidation (redox). Here, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its RPA32 and/or RPA14 subunits. Yeast RPA requires a reducing agent, such as dithiothreitol (DTT), for its ssDNA binding activity. Also, under non-reducing conditions, its DNA binding activity decreases 20 fold. In contrast, the RPA70 subunit does not require DTT for its DNA binding activity and is not affected by the redox condition. These results suggest that all three subunits are required for the regulation of RPA's DNA binding activity through redox potential.     

The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem–loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.     

RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions

The heterotrimeric DNA-binding protein, replication protein A (RPA), consists of 70-, 34-, and 14-kDa subunits and is involved in maintaining genomic stability by playing key roles in DNA replication, repair, and recombination. RPA participates in these processes through its interaction with other proteins and its strong affinity for single-stranded DNA (ssDNA). RPA-p34 is phosphorylated in a cell-cycle-dependent fashion primarily at Ser-29 and Ser-23, which are consensus sites for Cdc2 cyclin-dependent kinase. By systematically examining RPA-p34 phosphorylation throughout the cell cycle, we have found there are distinct phosphorylated forms of RPA-p34 in different cell-cycle stages. We have isolated and purified a unique phosphorylated form of RPA that is specifically associated with the mitotic phase of the cell cycle. The mitotic form of RPA (m-hRPA) shows no difference in ssDNA binding activity as compared with recombinant RPA (r-hRPA), yet binds less efficiently to double-stranded DNA (dsDNA). These data suggest that mitotic phosphorylation of RPA-p34 inhibits the destabilization of dsDNA by RPA complex, thereby decreasing the binding affinity for dsDNA. The m-hRPA also exhibits altered interactions with certain DNA replication and repair proteins. Using highly purified proteins, m-hRPA exhibited decreased binding to ATM, DNA pol alpha, and DNA-PK as compared to unphosphorylated recombinant RPA (r-hRPA). Dephosphorylation of m-hRPA was able to restore the interaction with each of these proteins. Interestingly, the interaction of RPA with XPA was not altered by RPA phosphorylation. These data suggest that phosphorylation of RPA-p34 plays an important role in regulating RPA functions in DNA metabolism by altering specific protein-protein interactions.     

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.     

DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage

Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS-PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.     

Replication protein A phosphorylation and the cellular response to DNA damage

Defects in cellular DNA metabolism have a direct role in many human disease processes. Impaired responses to DNA damage and basal DNA repair have been implicated as causal factors in diseases with DNA instability like cancer, Fragile X and Huntington's. Replication protein A (RPA) is essential for multiple processes in DNA metabolism including DNA replication, recombination and DNA repair pathways (including nucleotide excision, base excision and double-strand break repair). RPA is a single-stranded DNA-binding protein composed of subunits of 70-, 32- and 14-kDa. RPA binds ssDNA with high affinity and interacts specifically with multiple proteins. Cellular DNA damage causes the N-terminus of the 32-kDa subunit of human RPA to become hyper-phosphorylated. Current data indicates that hyper-phosphorylation causes a change in RPA conformation that down-regulates activity in DNA replication but does not affect DNA repair processes. This suggests that the role of RPA phosphorylation in the cellular response to DNA damage is to help regulate DNA metabolism and promote DNA repair.     

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.