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

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.

Download Full-text

APOBEC1 cytosine deaminase activity on single-stranded DNA is suppressed by replication protein A

Nucleic Acids Research ◽

10.1093/nar/gkaa1201 ◽

2020 ◽

Author(s):

Lai Wong ◽

Frederick S Vizeacoumar ◽

Franco J Vizeacoumar ◽

Linda Chelico

Keyword(s):

Genomic Dna ◽

Cytidine Deaminase ◽

Protein A ◽

Cytosine Deaminase ◽

Cancer Cell Line ◽

Replication Protein A ◽

Lung Cancer Cell Line ◽

Replication Protein ◽

Cancer Evolution

Abstract Many APOBEC cytidine deaminase members are known to induce ‘off-target’ cytidine deaminations in 5′TC motifs in genomic DNA that contribute to cancer evolution. In this report, we characterized APOBEC1, which is a possible cancer related APOBEC since APOBEC1 mRNA is highly expressed in certain types of tumors, such as lung adenocarcinoma. We found a low level of APOBEC1-induced DNA damage, as measured by γH2AX foci, in genomic DNA of a lung cancer cell line that correlated to its inability to compete in vitro with replication protein A (RPA) for ssDNA. This suggests that RPA can act as a defense against off-target deamination for some APOBEC enzymes. Overall, the data support the model that the ability of an APOBEC to compete with RPA can better predict genomic damage than combined analysis of mRNA expression levels in tumors and analysis of mutation signatures.

Download Full-text

Formation of a Complex between Nucleolin and Replication Protein a after Cell Stress Prevents Initiation of DNA Replication

The Journal of Cell Biology ◽

10.1083/jcb.149.4.799 ◽

2000 ◽

Vol 149 (4) ◽

pp. 799-810 ◽

Cited By ~ 80

Author(s):

Yaron Daniely ◽

James A. Borowiec

Keyword(s):

Dna Replication ◽

Ribosome Biogenesis ◽

Protein A ◽

Simian Virus 40 ◽

Replication Protein A ◽

Cell Stress ◽

Replication Protein ◽

Binding Studies

We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin–hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin–hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.

Download Full-text

Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro

Nucleic Acids Research ◽

10.1093/nar/gkg422 ◽

2003 ◽

Vol 31 (12) ◽

pp. 3006-3015 ◽

Cited By ~ 41

Author(s):

K.-H. Bae

Keyword(s):

Protein A ◽

Replication Protein A ◽

Replication Protein

Download Full-text

Novel Checkpoint Response to Genotoxic Stress Mediated by Nucleolin-Replication Protein A Complex Formation

Molecular and Cellular Biology ◽

10.1128/mcb.25.6.2463-2474.2005 ◽

2005 ◽

Vol 25 (6) ◽

pp. 2463-2474 ◽

Cited By ~ 56

Author(s):

Kyung Kim ◽

Diana D. Dimitrova ◽

Kristine M. Carta ◽

Anjana Saxena ◽

Mariza Daras ◽

...

Keyword(s):

Dna Replication ◽

Complex Formation ◽

Protein A ◽

Resonance Energy ◽

Genotoxic Stress ◽

Replication Protein A ◽

Replication Protein ◽

Chromosomal Dna

ABSTRACT Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.

Download Full-text

Functional Diversification of Replication Protein A Paralogs and Telomere Length Maintenance in Arabidopsis

Genetics ◽

10.1534/genetics.120.303222 ◽

2020 ◽

Vol 215 (4) ◽

pp. 989-1002

Author(s):

Behailu B. Aklilu ◽

François Peurois ◽

Carole Saintomé ◽

Kevin M. Culligan ◽

Daniela Kobbe ◽

...

Keyword(s):

Homologous Recombination ◽

Telomere Length ◽

Protein A ◽

Replication Protein A ◽

Telomerase Activity ◽

Replication Protein ◽

Telomeric Dna ◽

Telomere Shortening ◽

Replication Group

Replication protein A (RPA) is essential for many facets of DNA metabolism. The RPA gene family expanded in Arabidopsis thaliana with five phylogenetically distinct RPA1 subunits (RPA1A-E), two RPA2 (RPA2A and B), and two RPA3 (RPA3A and B). RPA1 paralogs exhibit partial redundancy and functional specialization in DNA replication (RPA1B and RPA1D), repair (RPA1C and RPA1E), and meiotic recombination (RPA1A and RPA1C). Here, we show that RPA subunits also differentially impact telomere length set point. Loss of RPA1 resets bulk telomeres at a shorter length, with a functional hierarchy for replication group over repair and meiosis group RPA1 subunits. Plants lacking RPA2A, but not RPA2B, harbor short telomeres similar to the replication group. Telomere shortening does not correlate with decreased telomerase activity or deprotection of chromosome ends in rpa mutants. However, in vitro assays show that RPA1B2A3B unfolds telomeric G-quadruplexes known to inhibit replications fork progression. We also found that ATR deficiency can partially rescue short telomeres in rpa2a mutants, although plants exhibit defects in growth and development. Unexpectedly, the telomere shortening phenotype of rpa2a mutants is completely abolished in plants lacking the RTEL1 helicase. RTEL1 has been implicated in a variety of nucleic acid transactions, including suppression of homologous recombination. Thus, the lack of telomere shortening in rpa2a mutants upon RTEL1 deletion suggests that telomere replication defects incurred by loss of RPA may be bypassed by homologous recombination. Taken together, these findings provide new insight into how RPA cooperates with replication and recombination machinery to sustain telomeric DNA.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.187.23.7881-7889.2005 ◽

2005 ◽

Vol 187 (23) ◽

pp. 7881-7889 ◽

Cited By ~ 11

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.

Download Full-text

Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing

Open Biology ◽

10.1098/rsob.110023 ◽

2011 ◽

Vol 1 (4) ◽

pp. 110023 ◽

Cited By ~ 30

Author(s):

Yuko Akai ◽

Yumiko Kurokawa ◽

Norihiko Nakazawa ◽

Yuko Tonami-Murakami ◽

Yuki Suzuki ◽

...

Keyword(s):

Protein A ◽

Coiled Coil ◽

Replication Protein A ◽

Replication Protein ◽

Mutation Site ◽

Damage Repair ◽

Dna Strands ◽

Structural Maintenance Of Chromosomes

Condensin is required for chromosome dynamics and diverse DNA metabolism. How condensin works, however, is not well understood. Condensin contains two structural maintenance of chromosomes (SMC) subunits with the terminal globular domains connected to coiled-coil that is interrupted by the central hinge. Heterotrimeric non-SMC subunits regulate SMC. We identified a novel fission yeast SMC hinge mutant, cut14-Y1 , which displayed defects in DNA damage repair and chromosome segregation. It contains an amino acid substitution at a conserved hinge residue of Cut14/SMC2, resulting in diminished DNA binding and annealing. A replication protein A mutant, ssb1-418 , greatly alleviated the repair and mitotic defects of cut14-Y1 . Ssb1 protein formed nucleolar foci in cut14-Y1 cells, but the number of foci was diminished in cut14-Y1 ssb1-418 double mutants. Consistent with the above results, Ssb1 protein bound to single-strand DNA was removed by condensin or the SMC dimer through DNA reannealing in vitro . Similarly, RNA hybridized to DNA may be removed by the SMC dimer. Thus, condensin may wind up DNA strands to unload chromosomal components after DNA repair and prior to mitosis. We show that 16 suppressor mutations of cut14-Y1 were all mapped within the hinge domain, which surrounded the original L543 mutation site.

Download Full-text

Replication protein A binds RNA and promotes R-loop formation

10.1101/2020.02.11.943977 ◽

2020 ◽

Author(s):

Olga M. Mazina ◽

Srinivas Somarowthu ◽

Lyudmila Y. Kadyrova ◽

Andrey G. Baranovskiy ◽

Tahir H. Tahirov ◽

...

Keyword(s):

Dna Replication ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Loop Formation ◽

Genome Maintenance ◽

Ssdna Binding ◽

Replication Restart ◽

Ssdna Binding Protein

SUMMARYReplication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA including DNA replication, repair, and damage signaling. Surprisingly, we found here that RPA binds RNA in vitro with high affinity. Using native RIP method, we isolated RNA-RPA complexes from human cells. Furthermore, RPA promotes R-loop formation between RNA and homologous dsDNA. R-loops, the three-stranded nucleic acid structure consisting of an RNA-DNA hybrid and the displaced ssDNA strand, are common in human genome. R-loops may play an important role in transcription-coupled homologous recombination and DNA replication restart. We reconstituted the process of replication restart in vitro using RPA-generated R-loops and human DNA polymerases. These findings indicate that RPA may play a role in RNA metabolism and suggest a mechanism of genome maintenance that depends on RPA and RNA.

Download Full-text