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.

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

1997 ◽  
Vol 17 (5) ◽  
pp. 2381-2390 ◽  
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.

Mutations in the Gene Encoding the 34 kDa Subunit of Yeast Replication Protein A Cause Defective S Phase Progression

1995 ◽  
Vol 254 (4) ◽  
pp. 595-607 ◽  
Author(s):  
Corrado Santocanale ◽  
Holger Neecke ◽  
Maria Pia Longhese ◽  
Giovanna Lucchini ◽  
Paolo Plevani
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein ◽  
Gene Encoding ◽  
Phase Progression

Phosphorylation of replication protein A by S-phase checkpoint kinases

DNA Repair ◽  
2006 ◽  
Vol 5 (3) ◽  
pp. 369-380 ◽  
Author(s):  
Jen-Sing Liu ◽  
Shu-Ru Kuo ◽  
Thomas Melendy
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein ◽  
Checkpoint Kinases ◽  
S Phase Checkpoint

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 Replication Protein A as a “Fidelity Clamp” for DNA Polymerase α

2001 ◽  
Vol 276 (21) ◽  
pp. 18235-18242 ◽  
Author(s):  
Giovanni Maga ◽  
Isabelle Frouin ◽  
Silvio Spadari ◽  
Ulrich Hübscher
Keyword(s):  
Dna Polymerase ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Dna Polymerase Α

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
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