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 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 Chl12 (Ctf18) Forms a Novel Replication Factor C-Related Complex and Functions Redundantly with Rad24 in the DNA Replication Checkpoint Pathway

2001 ◽  
Vol 21 (17) ◽  
pp. 5838-5845 ◽  
Author(s):  
Takahiro Naiki ◽  
Tae Kondo ◽  
Daisuke Nakada ◽  
Kunihiro Matsumoto ◽  
Katsunori Sugimoto
Keyword(s):  
Dna Replication ◽  
Dna Damage Checkpoint ◽  
Replication Factor C ◽  
Replication Checkpoint ◽  
Replication Factor ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
Replication Block ◽  
Factor C ◽  
Related Complex

ABSTRACT RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. Therad24Δ mutation enhances the defect ofrfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint.CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12Δ norrad24Δ single mutants are defective, chl12Δ rad24Δ double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.

scholarly journals ATAD5 deficiency alters DNA damage metabolism and sensitizes cells to PARP inhibition

2020 ◽  
Vol 48 (9) ◽  
pp. 4928-4939 ◽  
Author(s):  
Sara Giovannini ◽  
Marie-Christine Weller ◽  
Hana Hanzlíková ◽  
Tetsuya Shiota ◽  
Shunichi Takeda ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Parp Inhibitors ◽  
Parp Inhibition ◽  
Replication Forks ◽  
Replication Factor C ◽  
Knock Out ◽  
Endometrial Cancers ◽  
Factor C

Abstract Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.

scholarly journals Rfc5, a replication factor C component, is required for regulation of Rad53 protein kinase in the yeast checkpoint pathway.

1997 ◽  
Vol 17 (10) ◽  
pp. 5905-5914 ◽  
Author(s):  
K Sugimoto ◽  
S Ando ◽  
T Shimomura ◽  
K Matsumoto
Keyword(s):  
Dna Damage ◽  
Protein Kinase ◽  
Small Subunit ◽  
S Phase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Replication Factor C ◽  
Replication Factor ◽  
Checkpoint Pathway ◽  
Factor C

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae. We have previously shown that a temperature-sensitive (ts) rfc5-1 mutation is impaired in the S-phase checkpoint. In this report, we show that the rfc5-1 mutation is sensitive to DNA-damaging agents. RFC5 is necessary for slowing the S-phase progression in response to DNA damage. The phosphorylation of the essential central transducer, Rad53 protein kinase, is reduced in response to DNA damage in rfc5-1 mutants during the S phase. Furthermore, the inducibility of RNR3 transcription in response to DNA damage is dependent on RFC5. It has been shown that phosphorylation of Rad53 is controlled by Mec1 and Tel1, members of the subfamily of ataxia-telangiectasia mutated (ATM) kinases. We also demonstrate that overexpression of TEL1 suppresses the ts growth defect and DNA damage sensitivity of rfc5-1 mutants and restores phosphorylation of Rad53 and RNR3 induction in response to DNA damage in rfc5-1. Our results, together with the observation that overexpression of RAD53 suppresses the defects of the rfc5-1 mutation, suggest that Rfc5 is part of a mechanism transducing the DNA damage signal to the activation of the central transducer Rad53.

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.

Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability

1987 ◽  
Vol 7 (8) ◽  
pp. 2783-2793
Author(s):  
S J Elledge ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Small Subunit ◽  
Cross Reaction ◽  
Expression Library ◽  
Gene Encoding ◽  
Ribonucleotide Reductases ◽  
Colony Color

Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.

Upstream regulatory sequences of the yeast RNR2 gene include a repression sequence and an activation site that binds the RAP1 protein

1989 ◽  
Vol 9 (12) ◽  
pp. 5359-5372
Author(s):  
H K Hurd ◽  
J W Roberts
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribonucleotide Reductase ◽  
Small Subunit ◽  
Methyl Methanesulfonate ◽  
Regulatory Sequences ◽  
Start Site ◽  
Transcription Start ◽  
Dna Damaging Agents ◽  
Number Of Genes ◽  
Upstream Regulatory Sequences

The small subunit of ribonucleotide reductase in Saccharomyces cerevisiae (RNR2) was induced 3- to 20-fold by a variety of DNA-damaging agents. Induction of the RNR2 transcript by at least one of these agents, methyl methanesulfonate, did not require protein synthesis. To identify sequences involved in the regulation of RNR2, we introduced deletions upstream of the transcription start site. Sequences required for induction were contained within a 200-base-pair region that could confer methyl methanesulfonate inducibility on the heterologous CYC1 promoter. This region contained a repression sequence and at least two positive activation sites. One of these activation sites bound RAP1, a protein known to associate with mating-type silencers and the upstream activation sequences of a number of genes. The behavior of deletions of the repression sequence suggests that induction of RNR2 may occur, at least in part, through relief of repression.

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