scholarly journals Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA.

1988 ◽  
Vol 85 (8) ◽  
pp. 2523-2527 ◽  
Author(s):  
M. S. Wold ◽  
T. Kelly
Keyword(s):  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Simian Virus ◽  
Cellular Protein ◽  
Replication Protein ◽  
Purification And Characterization ◽  
In Vitro Replication

scholarly journals An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.

1995 ◽  
Vol 15 (10) ◽  
pp. 5396-5402 ◽  
Author(s):  
L Li ◽  
X Lu ◽  
C A Peterson ◽  
R J Legerski
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Simian Virus ◽  
Replication Protein ◽  
Nucleotide Excision

Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential.

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

2000 ◽  
Vol 149 (4) ◽  
pp. 799-810 ◽  
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.

scholarly journals The Middle Subunit of Replication Protein A Contacts Growing RNA-DNA Primers in Replicating Simian Virus 40 Chromosomes

1998 ◽  
Vol 18 (11) ◽  
pp. 6399-6407 ◽  
Author(s):  
Gilad Mass ◽  
Tamar Nethanel ◽  
Gabriel Kaufmann
Keyword(s):  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Simian Virus ◽  
Replication Protein ◽  
Specific Contact ◽  
Dynamic Attributes ◽  
Dna Primers ◽  
Lagging Strand

ABSTRACT The eukaryotic single-stranded DNA binding protein replication protein A (RPA) participates in major DNA transactions. RPA also interacts through its middle subunit (Rpa2) with regulators of the cell division cycle and of the response to DNA damage. A specific contact between Rpa2 and nascent simian virus 40 DNA was revealed by in situ UV cross-linking. The dynamic attributes of the cross-linked DNA, namely, its size distribution, RNA primer content, and replication fork polarity, were determined. These data suggest that Rpa2 contacts the early DNA chain intermediates synthesized by DNA polymerase α-primase (RNA-DNA primers) but not more advanced products. Possible signaling functions of Rpa2 are discussed, and current models of eukaryotic lagging-strand DNA synthesis are evaluated in view of our results.

scholarly journals Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.

1997 ◽  
Vol 17 (7) ◽  
pp. 3876-3883 ◽  
Author(s):  
C Iftode ◽  
J A Borowiec
Keyword(s):  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Simian Virus ◽  
T Antigen ◽  
Replication Protein ◽  
Origin Of Replication ◽  
Sv40 T Antigen ◽  
Viral Origin ◽  
Single Stranded Dna

The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.

scholarly journals Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

2004 ◽  
Vol 24 (5) ◽  
pp. 1930-1943 ◽  
Author(s):  
Vitaly M. Vassin ◽  
Marc S. Wold ◽  
James A. Borowiec
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Replication Protein ◽  
Wild Type ◽  
Chromosomal Dna

ABSTRACT Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2D again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2D. In contrast, under DNA damage or replication stress conditions, RPA2D, like RPA2A and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with γ-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.

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