To counter the threat of genomic damage, an evolutionarily conserved checkpoint system exists that recognizes the presence of damaged DNA, prevents cell cycle progression, and promotes repair. We were interested in understanding the mechanisms of (I) checkpoint initiation and (II) checkpoint termination during adaptation in Saccharomyces cerevisiae. The 9-1-1 clamp is a checkpoint sensor that is recruited to double-strand breaks (DSBs). Regarding checkpoint initiation, we examined the both the generic requirements and the recruitment patterns of the 9-1-1 checkpoint clamp to an engineered DSB. We discovered that while the 9-1-1 clamp shares structural and mechanical similarities with the PCNA-replication clamp to associate with DNA at ssDNA/dsDNA junctions, the genetic requirements for in vivo recruitment varied. Both clamp structures required the single-stranded binding protein complex, RPA. However, the 9-1-1 complex did not utilize the replication Polα-primase complex, which creates the ssDNA/dsDNA junctions recognized by PCNA. These data suggested the functional difference between the checkpoint and replication clamps lies in the substrate specificity. Regarding our interest in adaptation, we determined how the Cdc5 polo-like kinase acts to promote adaptation. Adaptation is a survival mechanism, in which yeast cells will escape a checkpoint arrest if DNA damage has not been repaired after several hours, and has been previously shown to require Cdc5. The overexpression of Cdc5 was used as a tool to probe how Cdc5 impacts checkpoint signaling. We found that Cdc5 overproduction had no significant effect on initial steps of checkpoint signaling, including recruitment of checkpoint sensors to damage and activity of initiating checkpoint kinases. However, the downstream checkpoint-effector kinase, Rad53, lost its damage-dependent hyperphosphorylation, suggesting Cdc5 may inhibit the amplification step of the checkpoint-signaling cascade.
|Advisor:||Toczyski, David P.|
|Commitee:||Li, Joachim J., Madhani, Hiten D.|
|School:||University of California, San Francisco|
|School Location:||United States -- California|
|Source:||DAI-B 70/04, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Adaptation, CDC5, DNA damage checkpoint, Polo-like kinase|
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