James A. Borowiec

Replication Protein A (RPA): The Eukaryotic SSB

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is highly conserved in eukaryotes. RPA plays essential roles in many aspects of nucleic acid metabolism, including DNA replication, nucleotide excision repair, and homologous recombination. In this review, we provide a comprehensive overview of RPA structure and function and highlight the more recent developments in these areas. The last few years have seen major advances in our understanding of the mechanism of RPA binding to DNA, including the structural characterization of the primary DNA-binding domains (DBD) and the identification of two secondary DBDs. Moreover, evidence indicates that RPA utilizes a multistep pathway to bind single-stranded DNA involving a particular molecular polarity of RPA, a mechanism that is apparently used to facilitate origin denaturation. In addition to its mechanistic roles, RPA interacts with many key factors in nucleic acid metabolism, and we discuss the critical nature of many of these interactions to DNA metabolism. RPA is a phosphorylation target for DNA-dependent protein kinase (DNA-PK) and likely the ataxia telangiectasia-mutated gene (ATM) protein kinase, and recent observations are described that suggest that RPA phosphorylation plays a significant modulatory role in the cellular response to DNA damage.

Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

ABSTRACT Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2D again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2D. In contrast, under DNA damage or replication stress conditions, RPA2D, like RPA2A and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with γ-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.

Stress-Dependent Nucleolin Mobilization Mediated by p53-Nucleolin Complex Formation

ABSTRACT We recently discovered that heat shock causes nucleolin to relocalize from the nucleolus to the nucleoplasm, whereupon it binds replication protein A and inhibits DNA replication initiation. We report that nucleolin mobilization also occurs following exposure to ionizing radiation (IR) and treatment with camptothecin. Mobilization was selective in that another nucleolar marker, upstream binding factor, did not relocalize in response to IR. Nucleolin relocalization was dependent on p53 and stress, the latter initially stimulating nucleolin-p53 complex formation. Nucleolin relocalization and complex formation in vivo were independent of p53 transactivation but required the p53 C-terminal regulatory domain. Nucleolin and p53 also interact directly in vitro, with a similar requirement for p53 domains. These data indicate a novel p53-dependent mechanism in which cell stress mobilizes nucleolin for transient replication inhibition and DNA repair.

Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA Repair

The activity of human replication protein A (RPA) in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are modified in vivo and in response to genotoxic stress. Using a novel antibody against phospho-Ser29, a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in nonstressed cells. Robust phosphorylation of Ser29 was also seen in interphase cells following treatment with the DNA-damaging agent camptothecin, a rare example of stress stimulating the modification of a repair factor by cyclin-Cdk. RPA2 phosphorylation is regulated both in cis and trans. Cis-phosphorylation follows a preferred pathway. (That is, the initial modification of Ser33 by ATR stimulates subsequent phosphorylation of Cdk sites Ser23 and Ser29). These events then facilitate modification of Thr21 and extreme N-terminal sites Ser4 and Ser8, probably by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans. Cells in which endogenous RPA2 was “replaced” with a double S23A/S29A-RPA2 mutant were seen to have an abnormal cell cycle distribution both in normal and in stressed cells. Such cells also showed aberrant DNA damage-dependent RPA foci and had persistent staining of γH2AX following DNA damage. Our data indicate that RPA phosphorylation facilitates chromosomal DNA repair. We postulate that the RPA phosphorylation pattern provides a means to regulate the DNA repair pathway utilized.

Single-stranded-DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase.

Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA8nt) and with 30 nucleotides (hRPA30nt). We find the relatively unstable hRPA8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.

A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination

Double-stranded DNA breaks (DSBs) induce a phosphorylation-mediated signaling cascade, but the role of phosphatases in this pathway remains unclear. Here we show that human protein phosphatase 4 (PP4) dephosphorylates replication protein A (RPA) subunit RPA2, regulating its role in the DSB response. PP4R2, a regulatory subunit of PP4, mediates the DNA damage–dependent association between RPA2 and the PP4C catalytic subunit. PP4 efficiently dephosphorylates phospho-RPA2 in vitro, and silencing PP4R2 in cells alters the kinetics and pattern of RPA2 phosphorylation. Depletion of PP4R2 impedes homologous recombination (HR) via inefficient loading of the essential HR factor RAD51, causing an extended G2-M checkpoint and hypersensitivity to DNA damage. Cells expressing phosphomimetic RPA2 mutants have a comparable phenotype, suggesting that PP4-mediated dephosphorylation of RPA2 is necessary for an efficient DNA-damage response. These observations provide new insight into the role and regulation of RPA phosphorylation in HR-mediated repair.

Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation.

ABSTRACT Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.

Nucleolin inhibits Hdm2 by multiple pathways leading to p53 stabilization

Nucleolin is a c-Myc-induced gene product with defined roles in ribosomal RNA processing and the inhibition of chromosomal DNA replication following stress. Here we find that changes in nucleolin protein levels in unstressed cells cause parallel changes in the amount of p53 protein. Alterations in p53 levels arise from nucleolin binding to the p53 antagonist Hdm2, resulting in the inhibition of both p53 ubiquitination and Hdm2 auto-ubiquitination. Nucleolin does not alter p53 ubiquitination by human papillomavirus E6, indicating that the effect is specific for Hdm2. Although the inhibition of ligase activity would be expected to stabilize Hdm2, we instead find that nucleolin also reduces Hdm2 protein levels, demonstrating that nucleolin inhibits Hdm2 using multiple mechanisms. Increases in nucleolin levels in unstressed cells led to higher expression of p21cip1/waf1, a reduced rate of cellular proliferation, and an increase in apoptosis. Thus, nucleolin has a number of properties in common with the tumor suppressor ARF (alternate reading frame). We propose that nucleolin, like ARF, responds to hyperproliferative signals by upregulation of p53 through Hdm2 inhibition.

Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery

Phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress.

RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage

Human replication protein A (RPA) becomes phosphorylated on the RPA2 subunit by cyclin B-Cdc2 during mitosis, although the functional role of this modification is unclear. We find that this modification stimulates RPA2 to become hyperphosphorylated in response to mitotic DNA damage caused by bleomycin treatment. Cells in which endogenous RPA2 was replaced by a mutant subunit lacking both Cdc2 sites had a significant defect in mitotic release into a 2N G1 phase after exposure to bleomycin. An increased percentage of these mutant cells also was positive initially for cyclin B expression and BubR1 chromatin staining, indicative of an extended spindle assembly checkpoint. The mutant cells that experienced mitotic DNA damage also underwent apoptosis at higher levels than cells expressing the WT subunit. Even so, we did not find the mutation had any dramatic effects on the level of DNA repair in mitosis. Cells lacking ATM (a checkpoint factor and RPA2 kinase) also were severely defective in mitotic exit and were unable to support RPA hyperphosphorylation after mitotic DNA damage. Although checkpoint 1 effector kinase (Chk1) had a more complex role, inhibition of Chk1 activity with UCN-01 also reduced mitotic exit. Chk1 activation and mitotic RPA hyperphosphorylation were found to be independent events. Our results demonstrate that mitotic RPA hyperphosphorylation facilitates release of cells from a damaged mitosis into a 2N G1 phase, thereby increasing cell viability.