Phosphorylation of replication protein A by S-phase checkpoint kinases

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.

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Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts

Nucleic Acids Research ◽

10.1093/nar/gkr1241 ◽

2011 ◽

Vol 40 (8) ◽

pp. 3431-3442 ◽

Cited By ~ 15

Author(s):

Bénédicte Recolin ◽

Siem Van Der Laan ◽

Domenico Maiorano

Keyword(s):

Protein A ◽

Replication Protein A ◽

S Phase ◽

Replication Protein ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

S Phase Checkpoint

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The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2381 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2381-2390 ◽

Cited By ~ 27

Author(s):

A E Parker ◽

R K Clyne ◽

A M Carr ◽

T J Kelly

Keyword(s):

Dna Repair ◽

Dna Synthesis ◽

Schizosaccharomyces Pombe ◽

Excision Repair ◽

Protein A ◽

Simian Virus 40 ◽

Replication Protein A ◽

S Phase ◽

Replication Protein

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

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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

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.3086-3096.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 3086-3096 ◽

Cited By ~ 188

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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