Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication

Replication protein A (RPA) plays an essential role in DNA replication by binding and unfolding non-canonical single-stranded DNA (ssDNA) structures. Of the six RPA ssDNA binding domains (labeled A-F), RPA-CDE selectively binds a G-quadruplex forming sequence (5′-TAGGGGAAGGGTTGGAGTGGGTT-3′called Gq23). In K+, Gq23 forms a mixed parallel/antiparallel conformation, and in Na+Gq23 has a less stable (TMlowered by ∼20∘C), antiparallel conformation. Gq23 is intramolecular and 1D NMR confirms a stable G-quadruplex structure in K+. Full-length RPA and RPA-CDE-core can bind and unfold the Na+form of Gq23 very efficiently, but complete unfolding is not observed with the K+form. Studies with G-quadruplex ligands, indicate that TMPyP4 has a thermal stabilization effect on Gq23 in K+, and inhibits complete unfolding by RPA and RPA-CDE-core. Overall these data indicate that G-quadruplexes present a unique problem for RPA to unfold and ligands, such as TMPyP4, could possibly hinder DNA replication by blocking unfolding by RPA.

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

The Cellular Response to DNA Damage Induced by the Enediynes C-1027 and Neocarzinostatin Includes Hyperphosphorylation and Increased Nuclear Retention of Replication Protein A (RPA) and Trans Inhibition of DNA Replication†

Biochemistry ◽

10.1021/bi001668t ◽

2001 ◽

Vol 40 (15) ◽

pp. 4792-4799 ◽

Cited By ~ 8

Author(s):

Mary M. McHugh ◽

Xia Yin ◽

Shu-Ru Kuo ◽

Jen-Sing Liu ◽

Thomas Melendy ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Cellular Response ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Nuclear Retention

Download Full-text

Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

Journal of Virology ◽

10.1128/jvi.78.4.1605-1615.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1605-1615 ◽

Cited By ~ 68

Author(s):

Yueh-Ming Loo ◽

Thomas Melendy

Keyword(s):

Dna Replication ◽

Protein A ◽

Replication Protein A ◽

T Antigen ◽

Replication Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Physical Interaction ◽

Single Stranded Dna ◽

Ssdna Binding ◽

Papillomavirus Dna

ABSTRACT With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.

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