Zn2+-Dependent Single-Stranded DNA Binding and DNA Replication Activities in Replication Protein A

ABSTRACT The replication clamp PCNA is loaded around DNA by replication factor C (RFC) and functions in DNA replication and repair. Regulated unloading of PCNA during the progression and termination of DNA replication may require additional factors. Here we show that a Saccharomyces cerevisiae complex required for the establishment of sister chromatid cohesion functions as an efficient unloader of PCNA. Unloading requires ATP hydrolysis. This seven-subunit Ctf18-RFC complex consists of the four small subunits of RFC, together with Ctf18, Dcc1, and Ctf8. Ctf18-RFC was also a weak loader of PCNA onto naked template-primer DNA. However, when the single-stranded DNA template was coated by the yeast single-stranded DNA binding protein replication protein A (RPA) but not by a mutant form of RPA or a heterologous single-stranded DNA binding protein, both binding of Ctf18-RFC to substrate DNA and loading of PCNA were strongly inhibited, and unloading predominated. Neither yeast RFC itself nor two other related clamp loaders, containing either Rad24 or Elg1, catalyzed significant unloading of PCNA. The Dcc1 and Ctf8 subunits of Ctf18-RFC, while required for establishing sister chromatid cohesion in vivo, did not function specifically in PCNA unloading in vitro, thereby separating the functionality of the Ctf18-RFC complex into two distinct paths.

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The RPA32 Subunit of Human Replication Protein A Contains a Single-stranded DNA-binding Domain

Journal of Biological Chemistry ◽

10.1074/jbc.273.7.3932 ◽

1998 ◽

Vol 273 (7) ◽

pp. 3932-3936 ◽

Cited By ~ 59

Author(s):

Elena Bochkareva ◽

Lori Frappier ◽

Aled M. Edwards ◽

Alexey Bochkarev

Keyword(s):

Dna Binding ◽

Protein A ◽

Replication Protein A ◽

Binding Domain ◽

Replication Protein ◽

Dna Binding Domain ◽

Single Stranded Dna

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Binding properties of replication protein A from human and yeast cells

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3050-3059.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3050-3059 ◽

Cited By ~ 1

Author(s):

C Kim ◽

R O Snyder ◽

M S Wold

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Protein A ◽

Simian Virus 40 ◽

Replication Protein A ◽

Simian Virus ◽

Yeast Cells ◽

Origin Of Replication ◽

Binding Properties ◽

Single Stranded Dna

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.

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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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Meiosis-specific proteins MEIOB and SPATA22 cooperatively associate with the single-stranded DNA-binding replication protein A complex and DNA double-strand breaks†

Biology of Reproduction ◽

10.1093/biolre/iox040 ◽

2017 ◽

Vol 96 (5) ◽

pp. 1096-1104 ◽

Cited By ~ 19

Author(s):

Yang Xu ◽

Roger A. Greenberg ◽

Ernst Schonbrunn ◽

P. Jeremy Wang

Keyword(s):

Dna Binding ◽

Protein A ◽

Replication Protein A ◽

Double Strand Breaks ◽

Replication Protein ◽

Dna Double Strand Breaks ◽

Single Stranded Dna ◽

Strand Breaks ◽

Specific Proteins

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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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FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA-binding protein

Blood ◽

10.1182/blood-2006-11-057273 ◽

2007 ◽

Vol 110 (7) ◽

pp. 2390-2398 ◽

Cited By ~ 74

Author(s):

Rigu Gupta ◽

Sudha Sharma ◽

Joshua A. Sommers ◽

Mark K. Kenny ◽

Sharon B. Cantor ◽

...

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Binding Protein ◽

Critical Role ◽

Protein A ◽

Replication Protein A ◽

Dna Binding Protein ◽

Replication Protein ◽

Single Stranded Dna

The BRCA1 associated C-terminal helicase (BACH1, designated FANCJ) is implicated in the chromosomal instability genetic disorder Fanconi anemia (FA) and hereditary breast cancer. A critical role of FANCJ helicase may be to restart replication as a component of downstream events that occur during the repair of DNA cross-links or double-strand breaks. We investigated the potential interaction of FANCJ with replication protein A (RPA), a single-stranded DNA-binding protein implicated in both DNA replication and repair. FANCJ and RPA were shown to coimmunoprecipitate most likely through a direct interaction of FANCJ and the RPA70 subunit. Moreover, dependent on the presence of BRCA1, FANCJ colocalizes with RPA in nuclear foci after DNA damage. Our data are consistent with a model in which FANCJ associates with RPA in a DNA damage-inducible manner and through the protein interaction RPA stimulates FANCJ helicase to better unwind duplex DNA substrates. These findings identify RPA as the first regulatory partner of FANCJ. The FANCJ-RPA interaction is likely to be important for the role of the helicase to more efficiently unwind DNA repair intermediates to maintain genomic stability.

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Complex formation between p53 and replication protein A inhibits the sequence-specific DNA binding of p53 and is regulated by single-stranded DNA.

Molecular and Cellular Biology ◽

10.1128/mcb.17.4.2194 ◽

1997 ◽

Vol 17 (4) ◽

pp. 2194-2201 ◽

Cited By ~ 28

Author(s):

S D Miller ◽

K Moses ◽

L Jayaraman ◽

C Prives

Keyword(s):

Dna Binding ◽

Complex Formation ◽

Binding Protein ◽

Protein A ◽

Replication Protein A ◽

Dna Binding Protein ◽

Replication Protein ◽

Single Stranded Dna ◽

C Terminus ◽

P53 Response

Human replication protein A (RP-A) (also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. Potentially important to both these functions, it is also capable of complex formation with the tumor suppressor protein p53. Here we show that although p53 is unable to prevent RP-A from associating with a range of single-stranded DNAs in solution, RP-A is able to strongly inhibit p53 from functioning as a sequence-specific DNA binding protein when the two proteins are complexed. This inhibition, in turn, can be regulated by the presence of various lengths of single-stranded DNAs, as RP-A, when bound to these single-stranded DNAs, is unable to interact with p53. Interestingly, the lengths of single-stranded DNA capable of relieving complex formation between the two proteins represent forms that might be introduced through repair and replicative events. Increasing p53 concentrations can also overcome the inhibition by steady-state levels of RP-A, potentially mimicking cellular points of balance. Finally, it has been shown previously that p53 can itself be stimulated for site-specific DNA binding when complexed through the C terminus with short single strands of DNA, and here we show that p53 stays bound to these short strands even after binding a physiologically relevant site. These results identify a potential dual role for single-stranded DNA in the regulation of DNA binding by p53 and give insights into the p53 response to DNA damage.

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