Mec1 and Rad53 inhibit formation of single-stranded DNA at telomeres of Saccharomyces cerevisiae cdc13-1 mutants.
Here we examine the roles of budding-yeast checkpoint proteins in regulating degradation of dsDNA to ssDNA at unprotected telomeres (in Cdc13 telomere-binding protein defective strains). We find that Rad17, Mec3, as well as Rad24, members of the putative checkpoint clamp loader (Rad24) and sliding clamp (Rad17, Mec3) complexes, are important for promoting degradation of dsDNA in and near telomere repeats. We find that Mec1, Rad53, as well as Rad9, have the opposite role: they inhibit degradation. Downstream checkpoint kinases Chk1 and Dun1 play no detectable role in either promoting degradation or inhibiting it. These data suggest, first, that the checkpoint sliding clamp regulates and/or recruits some nucleases for degradation, and, second, that Mec1 activates Rad9 to activate Rad53 to inhibit degradation. Further analysis shows that Rad9 inhibits ssDNA generation by both Mec1/Rad53-dependent and -independent pathways. Exo1 appears to be targeted by the Mec1/Rad53-dependent pathway. Finally, analysis of double mutants suggests a minor role for Mec1 in promoting Rad24-dependent degradation of dsDNA. Thus, checkpoint proteins orchestrate carefully ssDNA production at unprotected telomeres.
Jia
Xindan
X
School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.
Weinert
Ted
T
Lydall
David
D
D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004247:DNA / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D009154:Mutation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D016615:Telomere / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors
C452219:Cdc13 protein, S cerevisiae; D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D034501:Telomere-Binding Proteins; C069930:rad9 protein; D004247:DNA; D011494:Protein Kinases; C104169:DUN1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; D000071877:Checkpoint Kinase 1; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I