Processing of DNA in replication, repair and recombination pathways in cells of all organisms requires the participation of at least one major single-stranded DNA (ssDNA)-binding protein This protein protects ssDNA from nucleolytic damage, prevents hairpin formation and blocks DNA reannealing until the processing pathway is successfully completed Many ssDNA-binding proteins interact physically and functionally with a variety of other DNA processing proteins These interactions are thought to temporally order and guide the parade of proteins that 'trade places' on the ssDNA, a model known as 'hand-off', as the processing pathway progresses How this hand-off mechanism works remains poorly understood Recent studies of the conserved eukaryotic ssDNA-binding protein replication protein A (RPA) suggest a novel mechanism by which proteins may trade places on ssDNA by binding to RPA and mediating conformation changes that alter the ssDNA-binding properties of RPA This article reviews the structure and function of RPA, summarizes recent studies of RPA in DNA replication and other DNA processing pathways, and proposes a general model for the role of RPA in protein-mediated hand-off

/pdf/a-dynamic-model-for-replication-protein-a-rpa-function-in-5dgg4gkz9j.pdf

A dynamic model for replication protein A (RPA) function in DNA processing pathways

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

/pdf/rpa-coated-single-stranded-dna-as-a-platform-for-post-4x8t6g42kn.pdf

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

Fast time scale dynamics of protein backbones: NMR relaxation methods, applications, and functional consequences.

Replication protein A (RPA) and RAD51 are DNA-binding proteins that help maintain genome stability during DNA replication. These proteins regulate nucleases, helicases, DNA translocases, and signaling proteins to control replication, repair, recombination, and the DNA damage response. Their different DNA-binding mechanisms, enzymatic activities, and binding partners provide unique functionalities that cooperate to ensure that the appropriate activities are deployed at the right time to overcome replication challenges. Here we review and discuss the latest discoveries of the mechanisms by which these proteins work to preserve genome stability, with a focus on their actions in fork reversal and fork protection.

/pdf/rpa-and-rad51-fork-reversal-fork-protection-and-genome-54gayq2662.pdf

RPA and RAD51: fork reversal, fork protection, and genome stability

Replication protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis.

/pdf/replication-protein-a-single-stranded-dna-s-first-responder-4lesfswps2.pdf

Replication protein A: single-stranded DNA's first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair.

We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.

https://www.embopress.org/doi/pdf/10.1038/sj.emboj.7601432

Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex

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.

/pdf/nmr-study-on-the-interaction-between-rpa-and-dna-decamer-4ysvucodn2.pdf

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

Single-stranded-DNA-binding proteins (SSBs) are required for numerous genetic processes ranging from DNA synthesis to the repair of DNA damage, each of which requires binding with high affinity to ssDNA of variable base composition. To gain insight into the mechanism of sequence-nonspecific binding of ssDNA, NMR chemical shift and 15N relaxation experiments were performed on an isolated ssDNA-binding domain (RPA70A) from the human SSB replication protein A. The backbone 13C, 15N, and 1H resonances of RPA70A were assigned for the free protein and the d-CTTCA complex. The binding-induced changes in backbone chemical shifts were used to map out the ssDNA-binding site. Comparison to results obtained for the complex with d-C5 showed that the basic mode of binding is independent of the ssDNA sequence, but that there are differences in the binding surfaces. Amide nitrogen relaxation rates (R1 and R2) and 1H–15N NOE values were measured for RPA70A in the absence and presence of d-CTTCA. Analysis of the data using the Model-Free formalism and spectral density mapping approaches showed that the structural changes in the binding site are accompanied by some significant changes in flexibility of the primary DNA-binding loops on multiple timescales. On the basis of these results and comparisons to related proteins, we propose that the mechanism of sequence-nonspecific binding of ssDNA involves dynamic remodeling of the binding surface.

Characterization of binding-induced changes in dynamics suggests a model for sequence-nonspecific binding of ssDNA by replication protein A

Letter to the Editor: 1H, 13C and 15N Assignments of Single-Stranded DNA Binding Domains from the 70 kDa Subunit of Human Replication Protein A

1H, 15N and 13C assignments of single-stranded DNA binding domains from the 70 kDa subunit of Human Replication Protein A

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