The role of p14 subunit of replication protein A in binding to 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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Interactions of human replication protein A with single-stranded DNA adducts

Biochemical Journal ◽

10.1042/bj20041151 ◽

2005 ◽

Vol 385 (2) ◽

pp. 519-526 ◽

Cited By ~ 19

Author(s):

Yiyong LIU ◽

Zhengguan YANG ◽

Christopher D. UTZAT ◽

Yu LIU ◽

Nicholas E. GEACINTOV ◽

...

Keyword(s):

Excision Repair ◽

Protein A ◽

Chemical Properties ◽

Replication Protein A ◽

Replication Protein ◽

Base Stacking ◽

Single Stranded Dna ◽

Protein Residues ◽

Nucleotide Excision

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-aminopyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)-cis-anti-BPDE-dG, we demonstrated that, on binding of RPA, the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8–9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases.

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Human replication protein A binds single-stranded DNA in two distinct complexes.

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.3993 ◽

1994 ◽

Vol 14 (6) ◽

pp. 3993-4001 ◽

Cited By ~ 86

Author(s):

L J Blackwell ◽

J A Borowiec

Keyword(s):

Protein A ◽

Dna Recombination ◽

Replication Protein A ◽

Replication Protein ◽

Single Stranded Dna ◽

Ssdna Binding ◽

Ssdna Binding Protein ◽

Eukaryotic Dna ◽

Gel Shift

Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.

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Human replication protein A binds single-stranded DNA in two distinct complexes

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.3993-4001.1994 ◽

1994 ◽

Vol 14 (6) ◽

pp. 3993-4001

Author(s):

L J Blackwell ◽

J A Borowiec

Keyword(s):

Protein A ◽

Dna Recombination ◽

Replication Protein A ◽

Replication Protein ◽

Single Stranded Dna ◽

Ssdna Binding ◽

Ssdna Binding Protein ◽

Eukaryotic Dna ◽

Gel Shift

Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.

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Role of Replication Protein A in Double Holliday Junction Dissolution Mediated by the BLM-Topo IIIα-RMI1-RMI2 Protein Complex

Journal of Biological Chemistry ◽

10.1074/jbc.m113.465609 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14221-14227 ◽

Cited By ~ 24

Author(s):

Xiaoyu Xue ◽

Steven Raynard ◽

Valeria Busygina ◽

Akhilesh K. Singh ◽

Patrick Sung

Keyword(s):

Protein A ◽

Replication Protein A ◽

Dna Binding Protein ◽

Replication Protein ◽

Holliday Junction ◽

Interaction Domain ◽

Single Stranded Dna ◽

Dissolution Reaction ◽

Double Holliday Junction

The conserved BTR complex, composed of the Bloom's syndrome helicase (BLM), topoisomerase IIIα, RMI1, and RMI2, regulates homologous recombination in favor of non-crossover formation via the dissolution of the double Holliday Junction (dHJ). Here we show enhancement of the BTR-mediated dHJ dissolution reaction by the heterotrimeric single-stranded DNA binding protein replication protein A (RPA). Our results suggest that RPA acts by sequestering a single-stranded DNA intermediate during dHJ dissolution. We provide evidence that RPA physically interacts with RMI1. The RPA interaction domain in RMI1 has been mapped, and RMI1 mutants impaired for RPA interaction have been generated. Examination of these mutants ascertains the significance of the RMI1-RPA interaction in dHJ dissolution. Our results thus implicate RPA as a cofactor of the BTR complex in dHJ dissolution.

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