Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA

Nature ◽  
1997 ◽  
Vol 385 (6612) ◽  
pp. 176-181 ◽  
Author(s):  
Alexey Bochkarev ◽  
Richard A. Pfuetzner ◽  
Aled M. Edwards ◽  
Lori Frappier
Keyword(s):  
Dna Binding ◽  
Protein A ◽  
Replication Protein A ◽  
Binding Domain ◽  
Replication Protein ◽  
Dna Binding Domain ◽  
Single Stranded Dna

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

1998 ◽  
Vol 273 (7) ◽  
pp. 3932-3936 ◽  
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

scholarly journals In vitro analysis of the zinc-finger motif in human replication protein A

1999 ◽  
Vol 337 (2) ◽  
pp. 311-317 ◽  
Author(s):  
Jiaowang DONG ◽  
Jang-Su PARK ◽  
Suk-Hee LEE
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Dna Polymerase ◽  
Protein A ◽  
Replication Protein A ◽  
Binding Domain ◽  
Replication Protein ◽  
Zinc Finger Motif ◽  
Zinc Finger Domain ◽  
Single Stranded Dna

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase α-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys → Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase δ activity, whereas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2–3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPA's zinc finger domain in its function.

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

1996 ◽  
Vol 16 (9) ◽  
pp. 4798-4807 ◽  
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.

scholarly journals 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 ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2390-2398 ◽  
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.

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

1997 ◽  
Vol 17 (4) ◽  
pp. 2194-2201 ◽  
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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