Thesis

Investigating the Cds1 interaction with Cdc24, an essential schizosaccharomyces pombe replication factor

In Schizosaccharomyces pombe, Cdc24 is required for the completion of S-phase. Many studies implicate Cdc24 in lagging strand DNA replication and in DNA repair. Interestingly, the DNA replication checkpoint kinase, Cdsl (hChk2), and the kinase-dead allele, Cdsl-kd, causes a dosage growth defect in cdc24 truncation mutants (cdc24-M38 and cdc24-Gl) when overexpressed from a medium strength nmt promoter. This defect has also been reported in mutant alleles of mcll+, which encodes the DNA polymerase-a accessory factor. We hypothesize that the defect in cdc24 is due to the endonuclease Mus81, which is regulated by Cdsl to prevent unscheduled DNA cleavage. Alternatively, the defect is due to the Rad2 (hFenl) endonuclease. I used a genetic approach to investigate the dosage growth defect in order to understand the the function of Cdc24 with respect to Cdsl. To test for the involvement of Mus81, Rad2, and other replication factors such as Pol3, the catalytic subunit of DNA polymerase-6, I overexpressed Cdsl, Cdsl-kd, or an empty plasmid in cdc24 mutants that contain a background mutation at the mus81+, rad2+, pol3+, or other loci. Consistent with my hypothesis, my spot assay analyses show that the Cdsl or Cdsl-kd dosage growth defect in cdc24 is Rad2- dependent, but not Mus81-dependent. Intriguingly, we also found that Cdsl or Cdsl-kd caused a dosage growth defect in pol3 single mutant cells. These results suggest that Cdc24, Pol-6, and Mcll have overlapping functions in DNA replication and DNA repair, possibly to promote activation of Cdsl during a replication fork stall.

Relationships

Items