Replication Protein A Interactions with DNA. 2. Characterization of Double-Stranded DNA-Binding/Helix-Destabilization Activities and the Role of the Zinc-Finger Domain in DNA Interactions†

Biochemistry ◽  
1999 ◽  
Vol 38 (13) ◽  
pp. 3974-3984 ◽  
Author(s):  
Ye Lao ◽  
Chang Geun Lee ◽  
Marc S. Wold
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Protein A ◽  
Replication Protein A ◽  
Zinc Finger Domain ◽  
Dna Interactions ◽  
Double Stranded Dna ◽  
Dna 2

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.

Characterization of the DNA Binding Activity of the ZFY Zinc Finger Domain

Biochemistry ◽  
2010 ◽  
Vol 49 (4) ◽  
pp. 679-686 ◽  
Author(s):  
Jennifer Grants ◽  
Erin Flanagan ◽  
Andrea Yee ◽  
Paul J. Romaniuk
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Binding Activity ◽  
Zinc Finger Domain ◽  
Dna Binding Activity

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 A CCCH Zinc Finger Conserved in a Replication Protein A Homolog Found in Diverse Euryarchaeotes

2005 ◽  
Vol 187 (23) ◽  
pp. 7881-7889 ◽  
Author(s):  
Yuyen Lin ◽  
Justin B. Robbins ◽  
Ernest K. D. Nyannor ◽  
Yi-Hsing Chen ◽  
Isaac K. O. Cann
Keyword(s):  
Zinc Finger ◽  
Deletion Mutant ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Terminal Deletion ◽  
Zinc Finger Domain ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

ABSTRACT We describe a CCCH type of zinc finger domain in a replication protein A (RPA) homolog found in members of different lineages of the Euryarchaeota, a subdomain of Archaea. The zinc finger is characterized by CX2CX8CX2H, where X is any amino acid. Using MacRPA3, a representative of this new group of RPA in Methanosarcina acetivorans, we made two deletion mutants: a C-terminal deletion mutant lacking the zinc finger and an N-terminal deletion mutant containing the zinc finger domain. Whereas the N-terminal deletion mutant contained zinc at a level comparable to the wild-type protein level, the C-terminal deletion mutant was devoid of zinc. We further created four different mutants of MacRPA3 by replacing each of the four invariable amino acids in the zinc finger with alanine. Each single mutation at an invariable position resulted in a protein containing less than 35% of the zinc found in the wild-type protein. Circular dichroism spectra suggested that although the mutation at the first cysteine resulted in minor perturbation of protein structure, mutations at the other invariable positions led to larger structural changes. All proteins harboring a mutation at one of the invariable positions bound to single-stranded DNA weakly, and this translated into reduced capacity to stimulate DNA synthesis by M. acetivorans DNA polymerase BI. By subjecting the protein and its mutants to oxidizing and reducing conditions, we demonstrated that ssDNA binding by MacRPA3 may be regulated by redox through the zinc finger. Thus, the zinc finger modules in euryarchaeal RPA proteins may serve as a means by which the function of these proteins is regulated in the cell.

Replication Protein A Interactions with DNA. 1. Functions of the DNA-Binding and Zinc-Finger Domains of the 70-kDa Subunit†

Biochemistry ◽  
1999 ◽  
Vol 38 (13) ◽  
pp. 3963-3973 ◽  
Author(s):  
André P. Walther ◽  
Xavier V. Gomes ◽  
Ye Lao ◽  
Chang Geun Lee ◽  
Marc S. Wold
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Zinc Finger Domains
Sign in / Sign up

Export Citation Format

Share Document