DNA Stimulates Mec1-mediated Phosphorylation of Replication Protein A

Bartrand, Amy J.; Iyasu, Dagmawi; Brush, George S.

doi:10.1074/jbc.m312353200

Cited by 30 publications

(33 citation statements)

References 55 publications

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“…Next, we determined the role of Dpb11 and DNA in RPA phosphorylation. In agreement with previous studies (9,41), only DNA-bound RPA is an efficient target for phosphorylation by Mec1 (Fig. 1C).…”

Section: Resultssupporting

confidence: 80%

“…In yeast, both the Rpa1 and Rpa2 subunits of RPA are phosphorylated by Mec1 in response to DNA damage (39). In vitro, these two RPA subunits are phosphorylated by Mec1; however, phosphorylation is greatly stimulated when the RPA is bound to ssDNA (40,41). Addition of the 9-1-1 clamp loaded onto DNA stimulates the Mec1-mediated phosphorylation of DNA-bound RPA even further (9).…”

Section: Resultsmentioning

confidence: 99%

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Yeast DNA Replication Protein Dpb11 Activates the Mec1/ATR Checkpoint Kinase

Navadgi-Patil

Burgers

2008

Journal of Biological Chemistry

107

105

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The Saccharomyces cerevisiae Mec1-Ddc2 protein kinase (human ATR-ATRIP) initiates a signal transduction pathway in response to DNA damage and replication stress to mediate cell cycle arrest. The yeast DNA damage checkpoint clamp Ddc1-Mec3-Rad17 (human Rad9-Hus1-Rad1: 9-1-1) is loaded around effector DNA and thereby activates Mec1 kinase. Dpb11 (Schizosaccharomyces pombe Cut5/Rad4 or human TopBP1) is an essential protein required for the initiation of DNA replication and has a role in checkpoint activation. In this study, we demonstrate that Dpb11 directly activates the Mec1 kinase in phosphorylating the downstream effector kinase Rad53 (human Chk1/2) and DNA bound RPA. However, DNA was not required for Dpb11 to function as an activator. Dpb11 and yeast 9-1-1 independently activate Mec1, but substantial synergism in activation was observed when both activators were present. Our studies suggest that Dpb11 and 9-1-1 may partially compensate for each other during yeast checkpoint function.Phosphatidylinositol kinase-like protein kinases function as sensors in response to genotoxic stress to initiate a series of phosphorylation events that lead to a slowing down of cell cycle progression and promoting DNA repair. Ataxia telangiectasia mutated and ATR 2 are the major protein kinases that initiate DNA damage checkpoint in mammalian cells, although this function is carried out by Mec1 and Tel1 kinases in yeast Saccharomyces cerevisiae and Rad3 and Tel1 in Schizosaccharomyces pombe (reviewed in Refs. 1-3). The Ddc2 (human ATRIP) subunit of the heterodimeric Mec1-Ddc2 complex protein is required for association of Mec1 with single-stranded DNA coated with the single-stranded DNAbinding protein RPA (4, 5).DNA damage is also recognized by another group of sensor proteins called the checkpoint clamp-clamp loader complex (reviewed in Refs. 6, 7). The yeast 9-1-1 checkpoint clamp is a heterotrimer of the Ddc1, Rad17, and Mec3 proteins and is structurally similar to the replication clamp proliferating cell nuclear antigen. The yeast clamp loader for 9-1-1 is Rad24-RFC, and it consists of the four small subunits of replication factor C (Rfc2-5) together with the Rad24 subunit, the ortholog of Rad17 in S. pombe and human. The checkpoint clamp and clamp loader are required for the DNA damage checkpoint. During the G 1 phase of the cell cycle, RPA-coated ssDNA gaps that are generated during repair of DNA damage, e.g. by the nucleotide excision repair machinery, serve as a loading site for both the 9-1-1 clamp and Mec1 (8). We have recently reconstituted this checkpoint machinery in vitro with purified proteins (9). A partial duplex DNA with a 5Ј-primer-template junction and with the ssDNA region coated with RPA forms an efficient substrate for the loading of 9-1-1 by Rad24-RFC in an ATP-dependent reaction (10). This complex specifically activates Mec1 to phosphorylate its downstream targets. Among these targets are the Rpa1 and Rpa2 subunits of RPA, the Rad24 subunit of the clamp loader, and the Ddc1 and Mec3 subunits of the clamp....

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Yeast DNA Replication Protein Dpb11 Activates the Mec1/ATR Checkpoint Kinase

Navadgi-Patil

Burgers

2008

Journal of Biological Chemistry

107

105

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“…Furthermore, functional PP5 was required for the nuclear focus formation and hyperphosphorylation of RPA. Taken together with previous reports that ATR regulates the localization as well as phosphorylation of RPA (6,8), our results suggest an important role of PP5 in the activation of the ATR-mediated checkpoint pathway.…”

Section: Discussionsupporting

confidence: 73%

Protein Phosphatase 5 Is Required for ATR-Mediated Checkpoint Activation

Zhang¹,

Bao

Furumai³

et al. 2005

Molecular and Cellular Biology

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In response to DNA damage or replication stress, the protein kinase ATR is activated and subsequently transduces genotoxic signals to cell cycle control and DNA repair machinery through phosphorylation of a number of downstream substrates. Very little is known about the molecular mechanism by which ATR is activated in response to genotoxic insults. In this report, we demonstrate that protein phosphatase 5 (PP5) is required for the ATR-mediated checkpoint activation. PP5 forms a complex with ATR in a genotoxic stressinducible manner. Interference with the expression or the activity of PP5 leads to impairment of the ATRmediated phosphorylation of hRad17 and Chk1 after UV or hydroxyurea treatment. Similar results are obtained in ATM-deficient cells, suggesting that the observed defect in checkpoint signaling is the consequence of impaired functional interaction between ATR and PP5. In cells exposed to UV irradiation, PP5 is required to elicit an appropriate S-phase checkpoint response. In addition, loss of PP5 leads to premature mitosis after hydroxyurea treatment. Interestingly, reduced PP5 activity exerts differential effects on the formation of intranuclear foci by ATR and replication protein A, implicating a functional role for PP5 in a specific stage of the checkpoint signaling pathway. Taken together, our results suggest that PP5 plays a critical role in the ATR-mediated checkpoint activation.

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“…Recent work has shown that RPA bound to ssDNA serves as a binding platform for ATR-and Mec1-containing complexes, thereby localizing these protein kinases to sites of DNA damage (Zou and Elledge 2003). In addition, we have recently shown that ssDNA stimulates RPA phosphorylation catalyzed by immunoprecipitates of Mec1 (Bartrand et al 2004). These results have provided a possible explanation for the induction of RPA phosphorylation in vivo by many different events, all of which involve alterations in DNA structure.…”

Section: H Omologous Recombination During Meiosismentioning

confidence: 99%

Evidence of Meiotic Crossover Control in Saccharomyces cerevisiae Through Mec1-Mediated Phosphorylation of Replication Protein A

et al. 2006

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Replication protein A (RPA) is the major single-stranded DNA-binding protein in eukaryotes, essential for DNA replication, repair, and recombination. During mitosis and meiosis in budding yeast, RPA becomes phosphorylated in reactions that require the Mec1 protein kinase, a central checkpoint regulator and homolog of human ATR. Through mass spectrometry and site-directed mutagenesis, we have now identified a single serine residue in the middle subunit of the RPA heterotrimer that is targeted for phosphorylation by Mec1 both in vivo and in vitro. Cells containing a phosphomimetic version of RPA generated by mutation of this serine to aspartate exhibit a significant alteration in the pattern of meiotic crossovers for specific genetic intervals. These results suggest a new function of Mec1 that operates through RPA to locally control reciprocal recombination.

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DNA Stimulates Mec1-mediated Phosphorylation of Replication Protein A

Cited by 30 publications

References 55 publications

Yeast DNA Replication Protein Dpb11 Activates the Mec1/ATR Checkpoint Kinase

Yeast DNA Replication Protein Dpb11 Activates the Mec1/ATR Checkpoint Kinase

Protein Phosphatase 5 Is Required for ATR-Mediated Checkpoint Activation

Evidence of Meiotic Crossover Control in Saccharomyces cerevisiae Through Mec1-Mediated Phosphorylation of Replication Protein A

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