Publication Date

2024

Document Type

Dissertation

Committee Members

Yong-jie Xu, M.D., Ph.D. (Advisor); Michael Leffak, Ph.D. (Committee Member); Shulin Ju, Ph.D. (Committee Member); Quan Zhong, Ph.D. (Committee Member); Michael Kemp, Ph.D. (Committee Member)

Degree Name

Doctor of Philosophy (PhD)

Abstract

DNA replication can be perturbed by various agents that slow or stall the replication forks, causing replication stress. If undetected, stressed forks may collapse, causing mutagenic DNA damage or cell death. In response to replication stress and DNA damage, the eukaryotic cell activates the DNA replication checkpoint (DRC) and DNA damage checkpoint (DDC) pathways to promote DNA synthesis, repair, and cell survival. The two cell cycle checkpoint pathways are controlled by the protein sensor kinases Rad3 (hATR/scMec1) and Tel1 (hATM/scTel1) in fission yeast, although Tel1 plays a minimal role in checkpoint functions. Rad3 and Tel1 belong to a family of phosphatidylinositol-3-kinase-related kinases (PIKKs), whose stability is regulated by the heterotrimeric TTT (Tel2-Tti1-Tti2) complex. The current model suggests that the TTT complex works with Hsp90 and R2TP complex in the co-translational maturation of all PIKKs for their proper folding and stability. We have previously reported a tel2-C307Y mutant with a moderately reduced Rad3 protein level (~60% of wild-type cells). This mutation eliminates Rad3 mediated signaling in the DRC pathway but moderately reduces signaling in the DDC pathway. This result suggests that Tel2 of the TTT complex may specifically regulate the DRC pathway. In this study, we investigated this possibility by taking a genetic approach to analyze the functions of Tti1, the largest subunit of the TTT complex. We randomly mutated the tti1 gene and integrated the mutations at the genomic locus by pop-in and pop-out recombination strategy. As a result, 100 primary tti1 mutants were successfully screened, based on their increased sensitivities to hydroxyurea (HU) which depletes cellular dNTPs and/or the DNA damaging agent methyl methanesulfonate (MMS). Preliminary characterization of the primary Tti1 mutants, based on their relative sensitivities to HU, MMS or both agents, led us to focus on a collection of 24 mutants. Among the 24 mutants, DNA sequencing identified and subsequentially confirmed 8 novel tti1 mutants, which displayed reduced or moderately reduced checkpoint signaling in the DRC (Mrc1/Cds1 phosphorylation) and DDC (Chk1 phosphorylation) pathways. Consistent with the checkpoint signaling defect, the majority of the 8 tti1 mutants displayed cell untimely torn (cut) phenotype after HU treatment, confirming the DRC defect. Two tti1 mutants were not investigated further in this study due to their similar defects in the DRC and DDC pathways as compared with other mutants. Further investigation showed that Rad3 was moderately reduced in all six tti1 mutants as in the tel2-C307Y mutant. This suggests that Tti1 and Tel2 function as a single unit. We then performed tetrad dissection after crossing the six tti1 mutants with the tel2-C307Y mutant. All six tti1 mutants were found synthetic lethal with the tel2-C307Y mutant, which is consistent with the notion that Tti1 and Tel2 work in a single functional unit. Unexpectedly, the phosphorylation of Mrc1 in the tti1-N18 mutant is significantly different from that in the tel2-C307Y mutant and other newly identified tti1 mutants. As mentioned above, in response to HU treatment, Mrc1 is phosphorylated by both Rad3 and Tel1 although only the Rad3-mediated phosphorylation at the two threonine residues T645 and T653 is required for the DRC signaling. We found that while the tti1-N18 mutation eliminates the Rad3 phosphorylation of Mrc1, the Tel1 phosphorylation of Mrc1 remains largely intact. This result shows that while the co-translational maturation of Rad3 is likely affected in the tti1-N18 mutant, the mutation does not affect the maturation and stability of Tel1, another member of the PIKKs family. We are currently investigating this possibility by monitoring the protein levels of all six fission yeast PIKKs in the tti1-N18 mutants. If the hypothesis is confirmed, the TTT complex may regulate the maturation and stability of PIKKs with substrate specificity. It will also provide therapeutic opportunities that specifically target the cellular pathway controlled by a single member of the PIKKs family. Based on these results, we hypothesize that the TTT complex may regulate the checkpoint signaling in a Rad3-independent mechanism and the TTT may specifically interact with each of the PIKKs for their maturation and stability. Further experiments are needed to investigate these hypotheses.

Page Count

124

Department or Program

Biomedical Sciences

Year Degree Awarded

2024

ORCID ID

0000-0002-6130-4230

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