Physical interaction between archaeal DNA repair helicase Hel308 and Replication Protein A (RPA)

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

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Mechanism of Dynamic Binding of Replication Protein A to ssDNA

10.1101/2020.03.16.994681 ◽

2020 ◽

Author(s):

Anupam Mondal ◽

Arnab Bhattacherjee

Keyword(s):

Dna Repair ◽

Dynamic Equilibrium ◽

Diffusion Mechanism ◽

Protein A ◽

Principal Component ◽

Replication Protein A ◽

Replication Protein ◽

Coarse Grained ◽

Dna Processing

AbstractReplication protein A (RPA) serves as hub protein inside eukaryotic cells, where it coordinates crucial DNA metabolic processes and activates the DNA-damage response system. A characteristic feature of its action is to associate with ssDNA intermediates before handing over them to downstream proteins. The length of ssDNA intermediates differs for different pathways. This means RPA must have mechanisms for selective processing of ssDNA intermediates based on their length, the knowledge of which is fundamental to elucidate when and how DNA repair and replication processes are symphonized. By employing extensive molecular simulations, we investigated the mechanism of binding of RPA to ssDNA of different lengths. We show that the binding involves dynamic equilibrium with a stable intermediate, the population of which increases with the length of ssDNA. The vital underlying factors are decoded through collective variable principal component analysis. It suggests a differently orchestrated set of interactions that define the action of RPA based on the sizes of ssDNA intermediates. We further estimated the association kinetics and probed the diffusion mechanism of RPA to ssDNA. RPA diffuses on short ssDNA through progressive ‘bulge’ formation. With long ssDNA, we observed a conformational change in ssDNA coupled with its binding to RPA in a cooperative fashion. Our analysis explains how the ‘short-lived,’ long ssDNA intermediates are processed quickly in vivo. The study thus reveals the molecular basis of several recent experimental observations related to RPA binding to ssDNA and provides novel insights into the RPA functioning in DNA repair and replication.Significance StatementDespite ssDNA be the common intermediate to all pathways involving RPA, how does the latter function differently in the DNA processing events such as DNA repair, replication, and recombination just based on the length of ssDNA intermediates remains unknown. The major hindrance is the difficulty in capturing the transient interactions between the molecules. Even attempts to crystallize RPA complexes with 32nt and 62nt ssDNA have yielded a resolved structure of only 25nt ssDNA wrapped with RPA. Here, we used a state-of-the-art coarse-grained protein-ssDNA model to unravel the detailed mechanism of binding of RPA to ssDNA. Our study illustrates the molecular origin of variations in RPA action during various DNA processing events depending on the length of ssDNA intermediates.

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The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7222-7231.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7222-7231

Author(s):

V F Liu ◽

D T Weaver

Keyword(s):

Dna Repair ◽

Ionizing Radiation ◽

Gamma Irradiation ◽

Ataxia Telangiectasia ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Inherited Disease ◽

Dna Replication And Repair ◽

In Cells

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.

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Replication Protein A Confers Structure-specific Endonuclease Activities to the XPF-ERCC1 and XPG Subunits of Human DNA Repair Excision Nuclease

Journal of Biological Chemistry ◽

10.1074/jbc.271.19.11047 ◽

1996 ◽

Vol 271 (19) ◽

pp. 11047-11050 ◽

Cited By ~ 119

Author(s):

Tsukasa Matsunaga ◽

Chi-Hyun Park ◽

Tadayoshi Bessho ◽

David Mu ◽

Aziz Sancar

Keyword(s):

Dna Repair ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Human Dna

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Equilibrium and Stop-Flow Kinetic Studies of Fluorescently Labeled DNA Substrates with DNA Repair Proteins XPA and Replication Protein A

Biochemistry ◽

10.1021/bi011041q ◽

2002 ◽

Vol 41 (1) ◽

pp. 131-143 ◽

Cited By ~ 10

Author(s):

Lilia M. Iakoucheva ◽

Randall K. Walker ◽

Ben van Houten ◽

Eric J. Ackerman

Keyword(s):

Dna Repair ◽

Protein A ◽

Kinetic Studies ◽

Replication Protein A ◽

Replication Protein ◽

Dna Repair Proteins ◽

Dna Substrates ◽

Stop Flow

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Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

Journal of Virology ◽

10.1128/jvi.78.4.1605-1615.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1605-1615 ◽

Cited By ~ 68

Author(s):

Yueh-Ming Loo ◽

Thomas Melendy

Keyword(s):

Dna Replication ◽

Protein A ◽

Replication Protein A ◽

T Antigen ◽

Replication Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Physical Interaction ◽

Single Stranded Dna ◽

Ssdna Binding ◽

Papillomavirus Dna

ABSTRACT With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.

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Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.94.14.7186 ◽

1997 ◽

Vol 94 (14) ◽

pp. 7186-7191 ◽

Cited By ~ 62

Author(s):

N. A. Abramova ◽

J. Russell ◽

M. Botchan ◽

R. Li

Keyword(s):

Dna Repair ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Dependent Manner ◽

Uv Damage

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

Molecular and Cellular Biology ◽

10.1128/mcb.16.9.4798 ◽

1996 ◽

Vol 16 (9) ◽

pp. 4798-4807 ◽

Cited By ~ 76

Author(s):

L J Blackwell ◽

J A Borowiec ◽

I A Mastrangelo

Keyword(s):

Protein Kinase ◽

Dna Binding ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Physical Interaction ◽

Binding Modes ◽

Dependent Protein Kinase ◽

Single Stranded Dna ◽

Dependent Protein

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.

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