scholarly journals A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

2008 ◽  
Vol 284 (9) ◽  
pp. 5807-5818 ◽  
Author(s):  
Darren E. Casteel ◽  
Shunhui Zhuang ◽  
Ying Zeng ◽  
Fred W. Perrino ◽  
Gerry R. Boss ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Mammalian Cells ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein

scholarly journals DNA Replication but Not Nucleotide Excision Repair Is Required for UVC-Induced Replication Protein A Phosphorylation in Mammalian Cells

2000 ◽  
Vol 20 (8) ◽  
pp. 2696-2705 ◽  
Author(s):  
Gregory Rodrigo ◽  
Sophie Roumagnac ◽  
Marc S. Wold ◽  
Bernard Salles ◽  
Patrick Calsou
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Nucleotide Excision Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Dependent Manner ◽  
Nucleotide Excision

ABSTRACT Exposure of mammalian cells to short-wavelength light (UVC) triggers a global response which can either counteract the deleterious effect of DNA damage by enabling DNA repair or lead to apoptosis. Several stress-activated protein kinases participate in this response, making phosphorylation a strong candidate for being involved in regulating the cellular damage response. One factor that is phosphorylated in a UVC-dependent manner is the 32-kDa subunit of the single-stranded DNA-binding replication protein A (RPA32). RPA is required for major cellular processes like DNA replication, and removal of DNA damage by nucleotide excision repair (NER). In this study we examined the signal which triggers RPA32 hyperphosphorylation following UVC irradiation in human cells. Hyperphosphorylation of RPA was observed in cells from patients with either NER or transcription-coupled repair (TCR) deficiency (A, C, and G complementation groups of xeroderma pigmentosum and A and B groups of Cockayne syndrome, respectively). This exclude both NER intermediates and TCR as essential signals for RPA hyperphosphorylation. However, we have observed that UV-sensitive cells deficient in NER and TCR require lower doses of UV irradiation to induce RPA32 hyperphosphorylation than normal cells, indicating that persistent unrepaired lesions contribute to RPA phosphorylation. Finally, the results of UVC irradiation experiments on nonreplicating cells and S-phase-synchronized cells emphasize a major role for DNA replication arrest in the presence of UVC lesions in RPA UVC-induced hyperphosphorylation in mammalian cells.

scholarly journals The Mre11 Complex Mediates the S-Phase Checkpoint through an Interaction with Replication Protein A

2007 ◽  
Vol 27 (17) ◽  
pp. 6053-6067 ◽  
Author(s):  
Erin Olson ◽  
Christian J. Nievera ◽  
Enbo Liu ◽  
Alan Yueh-Luen Lee ◽  
Longchuan Chen ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Protein A ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Initiation ◽  
Replication Protein ◽  
Direct Role ◽  
Origin Firing ◽  
S Phase Checkpoint

ABSTRACT The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.

scholarly journals Stabilization of a G-Quadruplex from Unfolding by Replication Protein A Using Potassium and the Porphyrin TMPyP4

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Aishwarya Prakash ◽  
Fabien Kieken ◽  
Luis A. Marky ◽  
Gloria E. O. Borgstahl
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Thermal Stabilization ◽  
Replication Protein A ◽  
Replication Protein ◽  
Unique Problem ◽  
Ssdna Binding ◽  
Binding Domains ◽  
G Quadruplex ◽  
Stabilization Effect

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.

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 Assembly of a Complex Containing Cdc45p, Replication Protein A, and Mcm2p at Replication Origins Controlled by S-Phase Cyclin-Dependent Kinases and Cdc7p-Dbf4p Kinase

2000 ◽  
Vol 20 (9) ◽  
pp. 3086-3096 ◽  
Author(s):  
Lee Zou ◽  
Bruce Stillman
Keyword(s):  
Dna Polymerase ◽  
Origin Recognition Complex ◽  
Protein A ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein ◽  
Dependent Manner ◽  
Replication Origins ◽  
Cyclin Dependent Kinases ◽  
Origin Activation

ABSTRACT In Saccharomyces cerevisiae, replication origins are activated with characteristic timing during S phase. S-phase cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required for origin activation throughout S phase. The activation of S-CDKs leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p at the G1-S transition in an S-CDK-dependent manner. During S phase, Cdc45p associates with different replication origins at specific times. The origin associations of Cdc45p and RPA are mutually dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins. These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing. TheARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element ofARS1, a potential origin DNA-unwinding element. Cdc45p is required for recruiting DNA polymerase α onto chromatin, and it associates with Mcm2p, RPA, and DNA polymerase ɛ only during S phase. These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at each origin.

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