GSBS Dissertations and Theses

Approval Date

5-21-2009

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program

Subjects

DNA Damage; DNA Helicases; DNA-Binding Proteins; Endonucleases; S Phase; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; RecQ Helicases; Academic Dissertations; Dissertations, UMMS

Abstract

Faithful duplication and segregation of undamaged DNA is critical to the survival of all organisms and prevention of oncogenesis in multicellular organisms. To ensure inheritance of intact DNA, cells rely on checkpoints. Checkpoints alter cellular processes in the presence of DNA damage preventing cell cycle transitions until replication is completed or DNA damage is repaired.

Several checkpoints are specific to S-phase. The S-M replication checkpoint prevents mitosis in the presence of unreplicated DNA. Rather than outright halting replication, the S-phase DNA damage checkpoint slows replication in response to DNA damage. This checkpoint utilizes two general mechanisms to slow replication. First, this checkpoint prevents origin firing thus limiting the number of replication forks traversing the genome in the presence of damaged DNA. Second, this checkpoint slows the progression of the replication forks. Inhibition of origin firing in response to DNA damage is well established, however when this thesis work began, slowing of replication fork progression was controversial.

Fission yeast slow replication in response to DNA damage utilizing an evolutionarily conserved kinase cascade. Slowing requires the checkpoint kinases Rad3 (hATR) and Cds1 (hChk2) as well as additional checkpoint components, the Rad9-Rad1-Hus1 complex and the Mre11-Rad50-Nbs1 (MRN) recombinational repair complex. The exact role MRN serves to slow replication is obscure due to its many roles in DNA metabolism and checkpoint response to damage. However, fission yeast MRN mutants display defects in recombination in yeast and, upon beginning this project, were described in vertebrates to display S-phase DNA damage checkpoint defects independent of origin firing.

Due to these observations, I initially hypothesized that recombination was required for replication slowing. However, two observations forced a paradigm shift in how I thought replication slowing to occur and how replication fork metabolism was altered in response to DNA damage. We found rhp51Δ mutants (mutant for the central mitotic recombinase similar to Rad51 and RecA) to slow well. We observed that the RecQ helicase Rqh1, implicated in negatively regulating recombination, was required for slowing. Therefore, deregulated recombination appeared to actually be responsible for slowing failures exhibited by the rqh1Δ recombination regulator mutant. Thereafter, I began a search for additional regulators required for slowing and developed the epistasis grouping described in Chapters II and V.

We found a wide variety of mutants which either completely or partially failed to slow replication in response to DNA damage. The three members of the MRN complex, nbs1Δ, rad32Δ and rad50Δ displayed a partial defect in slowing, as did the helicase rqh1Δ and Rhp51-mediator sfr1Δ mutants. We found the mus81Δ and eme1Δ endonuclease complex and the smc6-x hypomorph to completely fail to slow.

We were able to identify at least three epistasis groups due to genetic interaction between these mutants and recombinase mutants. Interestingly, not all mutants’ phenotypes were suppressed by abrogation of recombination. As introduced in Chapters II, III and IV checkpoint kinase cds1Δ, mus81Δ endonuclease, and smc6-x mutant slowing defects were not suppressed by abrogation of recombination, while the sfr1Δ, rqh1Δ, rad2Δ and nbs1Δ mutant slowing defects were.

Additionally, data shows replication slowing in fission yeast is primarily due to proteins acting locally at sites of DNA damage. We show that replication slowing is lesion density-dependent, prevention of origin firing representing a global response to insult contributes little to slowing, and constitutive checkpoint activation is not sufficient to induce DNA damage-independent slowing.

Collectively, our data strongly suggest that slowing of replication in response to DNA damage in fission yeast is due to the slowing of replication forks traversing damaged template. We show slowing must be primarily a local response to checkpoint activation and all mutants found to fail to slow are implicated in replication fork metabolism, and recombination is responsible for some mutant slowing defects.

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