scholarly journals Rfc4 Interacts with Rpa1 and Is Required for Both DNA Replication and DNA Damage Checkpoints in Saccharomyces cerevisiae

2001 ◽  
Vol 21 (11) ◽  
pp. 3725-3737 ◽  
Author(s):  
Hee-Sook Kim ◽  
Steven J. Brill
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Essential Role ◽  
Large Subunit ◽  
Small Subunit ◽  
Dna Damage Checkpoint ◽  
Physical Interaction ◽  
Clamp Loader ◽  
Synthesis Inhibitor ◽  
Factor C

ABSTRACT The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such asrfa1-t11, are lethal in combination withrfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint geneRAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G1/S DNA damage checkpoint response and that both therfc4-2 and rfa1-t11 strains are defective in the G2/M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.

scholarly journals Functional and Physical Interaction between Rad24 and Rfc5 in the Yeast Checkpoint Pathways

1998 ◽  
Vol 18 (9) ◽  
pp. 5485-5491 ◽  
Author(s):  
Toshiyasu Shimomura ◽  
Seiko Ando ◽  
Kunihiro Matsumoto ◽  
Katsunori Sugimoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Small Subunit ◽  
Dna Damage Checkpoint ◽  
Physical Interaction ◽  
Replication Factor C ◽  
Functional Interaction ◽  
Dna Damaging Agents ◽  
Replication Block ◽  
Factor C

ABSTRACT The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage. Here we describe the isolation ofRAD24, known to play a role in the DNA damage checkpoint, as a dosage-dependent suppressor of rfc5-1. RAD24overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation. Rad24, like Rfc5, is required for the regulation of Rad53 phosphorylation in response to DNA damage. The Rad24 protein, which is structurally related to the RFC subunits, interacts physically with RFC subunits Rfc2 and Rfc5 and cosediments with Rfc5. Although therad24Δ mutation alone does not cause a defect in the replication block checkpoint, it does enhance the defect inrfc5-1 mutants. Furthermore, overexpression ofRAD24 suppresses the rfc5-1 defect in the replication block checkpoint. Taken together, our results demonstrate a physical and functional interaction between Rad24 and Rfc5 in the checkpoint pathways.

scholarly journals The Large Subunit of Replication Factor C (Rfclp/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 65-78 ◽  
Author(s):  
Michael A McAlear ◽  
K Michelle Tuffo ◽  
Connie Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Dna Replication ◽  
Uv Radiation ◽  
Large Subunit ◽  
Restrictive Temperature ◽  
Replication Factor C ◽  
Replication Factor ◽  
Factor C

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.

scholarly journals The Yeast PCNA Unloader Elg1 RFC-Like Complex Plays a Role in Eliciting the DNA Damage Checkpoint

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Soumitra Sau ◽  
Batia Liefshitz ◽  
Martin Kupiec
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Cellular Response ◽  
Genome Stability ◽  
Adaptor Proteins ◽  
Nuclear Antigen ◽  
Dna Damage Checkpoint ◽  
Activation Process ◽  
Checkpoint Activation ◽  
Dna Replication And Repair

ABSTRACT The PCNA (proliferating cell nuclear antigen) ring plays central roles during DNA replication and repair. The yeast Elg1 RFC-like complex (RLC) is the principal unloader of chromatin-bound PCNA and thus plays a central role in maintaining genome stability. Here we identify a role for Elg1 in the unloading of PCNA during DNA damage. Using DNA damage checkpoint (DC)-inducible and replication checkpoint (RC)-inducible strains, we show that Elg1 is essential for eliciting the signal in the DC branch. In the absence of Elg1 activity, the Rad9 (53BP1) and Dpb11 (TopBP1) adaptor proteins are recruited but fail to be phosphorylated by Mec1 (ATR), resulting in a lack of checkpoint activation. The chromatin immunoprecipitation of PCNA at the Lac operator sites reveals that accumulated local PCNA influences the checkpoint activation process in elg1 mutants. Our data suggest that Elg1 participates in a mechanism that may coordinate PCNA unloading during DNA repair with DNA damage checkpoint induction. IMPORTANCE The Elg1protein forms an RFC-like complex in charge of unloading PCNA from chromatin during DNA replication and repair. Mutations in the ELG1 gene caused genomic instability in all organisms tested and cancer in mammals. Here we show that Elg1 plays a role in the induction of the DNA damage checkpoint, a cellular response to DNA damage. We show that this defect is due to a defect in the signal amplification process during induction. Thus, cells coordinate the cell's response and the PCNA unloading through the activity of Elg1.

scholarly journals Activation of the DNA Damage Checkpoint in Yeast Lacking the Histone Chaperone Anti-Silencing Function 1

2004 ◽  
Vol 24 (23) ◽  
pp. 10313-10327 ◽  
Author(s):  
Christopher Josh Ramey ◽  
Susan Howar ◽  
Melissa Adkins ◽  
Jeffrey Linger ◽  
Judson Spicer ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Histone Chaperone ◽  
Dna Damage Checkpoint ◽  
Genomic Integrity ◽  
Dna Breaks ◽  
Double Strand Dna

ABSTRACT The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of genomic integrity. Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin assembly factors, including anti-silencing function 1 (ASF1). To investigate the role of chromatin structure in the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p. We found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage checkpoint. Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and to DNA replicational stresses. Although yeast lacking Asf1p do complete DNA replication, they have greatly elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green fluorescent protein foci. The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are fully functional for double-strand DNA repair. Our data indicate that the altered chromatin structure in asf1 mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.

scholarly journals CDC44: a putative nucleotide-binding protein required for cell cycle progression that has homology to subunits of replication factor C.

1994 ◽  
Vol 14 (1) ◽  
pp. 255-267 ◽  
Author(s):  
E A Howell ◽  
M A McAlear ◽  
D Rose ◽  
C Holm
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Large Subunit ◽  
Mitotic Cell ◽  
Predicted Amino Acid Sequence ◽  
Replication Factor C ◽  
Replication Factor ◽  
Cycle Arrest ◽  
Factor C

To investigate the means by which a cell regulates the progression of the mitotic cell cycle, we characterized cdc44, a mutation that causes Saccharomyces cerevisiae cells to arrest before mitosis. CDC44 encodes a 96-kDa basic protein with significant homology to a human protein that binds DNA (PO-GA) and to three subunits of human replication factor C (also called activator 1). The hypothesis that Cdc44p is involved in DNA metabolism is supported by the observations that (i) levels of mitotic recombination suggest elevated rates of DNA damage in cdc44 mutants and (ii) the cell cycle arrest observed in cdc44 mutants is alleviated by the DNA damage checkpoint mutations rad9, mec1, and mec2. The predicted amino acid sequence of Cdc44p contains GTPase consensus sites, and mutations in these regions cause a conditional cell cycle arrest. Taken together, these observations suggest that the essential CDC44 gene may encode the large subunit of yeast replication factor C.

scholarly journals Regulation of RelA (p65) Function by the Large Subunit of Replication Factor C

2003 ◽  
Vol 23 (2) ◽  
pp. 721-732 ◽  
Author(s):  
Lisa A. Anderson ◽  
Neil D. Perkins
Keyword(s):  
Tumor Suppressor ◽  
Large Subunit ◽  
Dominant Negative ◽  
Nuclear Antigen ◽  
Replication Factor C ◽  
Clamp Loader ◽  
Replication Factor ◽  
Factor C

ABSTRACT The RelA (p65) subunit of NF-κB is an important regulator of inflammation, proliferation, and apoptosis. We have discovered that the large subunit, p140, of replication factor C (RFC) can function as a regulator of RelA. RFC is a clamp loader, facilitating the addition and removal of proliferating-cell nuclear antigen from DNA during replication and repair but can also interact directly with the retinoblastoma tumor suppressor protein and the transcription factor C/EBPα. We find that RFC (p140) interacts with RelA both in vitro and in vivo and stimulates RelA transactivation. In contrast, coexpression of fragments of RFC (p140) that mediate the interaction with RelA results in transcriptional inhibition. The significance of this regulation was confirmed by using short interfering RNA oligonucleotides targeted to RFC (p140). Down regulation of endogenous RFC (p140) inhibits expression from a chromosomally integrated reporter plasmid induced by endogenous, TNF-α-activated NF-κB. Dominant negative fragments of RFC (p140) also cooperate with overexpressed RelA to induce cell death. Interestingly, RFC (p140) also interacts with the tumor suppressor p53. Taken together, these observations suggest that, in addition to its previously described function in DNA replication and repair, RFC (p140) has an important role as a regulator of transcription and NF-κB activity.

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