In eukaryotic cells, DNA damage causes arrest of the cell cycle to allow repair of this damage. A number of genes, termed """"""""checkpoint"""""""" genes, required for this arrest have been identified in yeast and in other organisms. Human checkpoint genes include p53 and ATM and mutations in these genes are associated with increased probability of developing cancer. Dr. Weinert suggests that there may be three types of checkpoint proteins: 1) sensor proteins which bind and, perhaps, process DNA damage, 2) signal proteins that interact with the sensor protein and transduce a signal to target genes, and 3) the target genes, which directly mediate cell cycle arrest. His first series of experiments concern how sensor proteins act on DNA damage. Strains with the cdc13 mutation develop long single-stranded regions near the end of the chromosome. Dr. Weinert has found that mutations in the rad24 checkpoint gene reduced this degradation, and mutations in the rad9 checkpoint gene increased the degradation. He will determine whether the degradation occurs 3' to 5' or 5' to 3', and he will examine the effects of other mutations (for example, rad17) on the degradation process. The Rad17p shares homology with Rec1p, a known 3' to 5' exonuclease. Dr. Weinert will purify Rad17p and determine whether Rad17p is a 3' to 5' exonuclease. He will also try to localize Rad17p, Rad24 and Mec3p to sites of meiosis-specific DSB's using immunofluorescence. The point of these studies is to test the hypothesis that these proteins represent sensor proteins that process the cdc13-caused DNA damage. The second part of the proposal is designed to address two questions. First, is processing of DNA damage by Rad24p and Rad17p required to repair this DNA damage? Second, does cell cycle arrest require Rad24p and Rad17p to process the DNA damage? Dr. Weinert will try to answer these questions by mutating RAD17 and RAD24, and determining whether any of the mutant alleles show separation of function. For example, if he obtains a rad17 mutant that does not process DNA damage, but does show cell-cycle arrest, he will have demonstrated that DNA processing is not required for cell-cycle arrest. The third part of the proposal concerns new checkpoint mutants. His previous hunts involved looking for mutants that had poor viability after exposure to DNA damage. His new hunt will employ a direct search for cells that fail to show G2/M arrest in response to DNA damage. He hopes that this search will identify target genes of the checkpoint response. The last series of experiments investigate the order of function of genes in the checkpoint pathway. Dr. Weinert proposes looking at protein-protein interactions between different checkpoint proteins using the two-hybrid system or affinity columns. He also proposes two types of epistasis tests. Work from the Elledge lab suggests that the Rad53p is phosphorylated as part of the checkpoint response and functions downstream of Mec1p and the sensor proteins. Dr. Weinert will attempt to confirm this conclusion. If the conclusion is confirmed, he will determine the effect of various checkpoint mutations on the phosphorylation of Rad53p. He will also attempt to isolate a mec1 mutant with the phenotype of cold-sensitive constitutive function. He will then examine interactions with other mutants. A double mutant with a mec1 constitutive and a mutant in a gene that functions downstream of MEC1 should fail to show arrest at the restrictive temperature.

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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Arizona
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Admire, Anthony; Shanks, Lisa; Danzl, Nicole et al. (2006) Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast. Genes Dev 20:159-73
Weinert, T; Little, E; Shanks, L et al. (2000) Details and concerns regarding the G2/M DNA damage checkpoint in budding yeast. Cold Spring Harb Symp Quant Biol 65:433-41
Gardner, R; Putnam, C W; Weinert, T (1999) RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast. EMBO J 18:3173-85
Lydall, D; Weinert, T (1997) Use of cdc13-1-induced DNA damage to study effects of checkpoint genes on DNA damage processing. Methods Enzymol 283:410-24
Kim, S; Weinert, T A (1997) Characterization of the checkpoint gene RAD53/MEC2 in Saccharomyces cerevisiae. Yeast 13:735-45
Lydall, D; Weinert, T (1997) G2/M checkpoint genes of Saccharomyces cerevisiae: further evidence for roles in DNA replication and/or repair. Mol Gen Genet 256:638-51
Kiser, G L; Weinert, T A (1996) Distinct roles of yeast MEC and RAD checkpoint genes in transcriptional induction after DNA damage and implications for function. Mol Biol Cell 7:703-18
Lydall, D; Weinert, T (1995) Yeast checkpoint genes in DNA damage processing: implications for repair and arrest. Science 270:1488-91
Kiser, G L; Weinert, T A (1995) GUF1, a gene encoding a novel evolutionarily conserved GTPase in budding yeast. Yeast 11:1311-6
Weinert, T A; Kiser, G L; Hartwell, L H (1994) Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev 8:652-65

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