Date of Completion

9-18-2018

Embargo Period

9-17-2019

Keywords

DNA Mismatch repair, Replication stress response, DNA damage response, Alkylation damage

Major Advisor

Dr. Christopher D. Heinen

Associate Advisor

Dr. Ann E. Cowan

Associate Advisor

Dr. Gordon G. Carmichael

Associate Advisor

Dr. Stormy J. Chamberlain

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The DNA mismatch repair (MMR) pathway, a crucial post-replicative repair pathway, is essential to the maintenance of genomic stability. Consequentially, loss of MMR increases mutation frequency, promoting tumorigenesis. However, if and how MMR activity coordinates with cellular replication forks is unclear. Particularly, at forks encountering lesions that cannot be faithfully replicated, like O6-methylguanine (MeG) lesions created by DNA alkylating agents, MMR-directed processing of resultant mismatches could disrupt fork progression. Yet the events following lesion recognition remain elusive. In transformed cells MMR-dependent MeG/T recognition in the first S phase elicits a permanent G2 arrest in the subsequent cell cycle. Yet, in human pluripotent stem cells (hPSCs), it activates an immediate and robust MMR-dependent apoptotic response. In this study, we ascertained how MeG/T lesion recognition affects the first S phase in human embryonic stem cells (hESCs) and transformed HeLa cells. MMR-proficient HeLa cells exposed to alkylation damage activated ataxia telangiectasia and Rad3-related (ATR)-Checkpoint Kinase 1 (Chk1) signaling, slowing progression through the first S phase. Yet, DNA replication is completed, and cells progress into the next cell cycle. Conversely, inhibition of ATR-Chk1 signaling accelerates S phase progression, damage accumulation and sensitivity to DNA alkylating agents. In addition, as MMR-proficient human embryonic stem cells exposed to alkylation damage fail to activate ATR-Chk1 signaling, MMR activity severely compromises DNA replication leading to accumulation of toxic double strand breaks and rapid apoptotic induction. We propose that MMR-directed futile repair cycles disrupt fork progression, implicating the MMR-directed repair of alkylation damage as a replication stress inducer. Thereafter, ATR-Chk1 mediated intra-S phase checkpoint activation mitigates replication stress and facilitates completion of replication. Absence of this signaling, however, accelerates damage accumulation and sensitivity to alkylating agents. This work changes how we view this post-replicative repair pathway, suggesting a coordination between MMR activity and the DNA replication machinery. In addition, these results reveal that different cell types may have different levels of sensitivity to MMR processing of lesions. Taken together, this work provides the foundation for understanding how MMR is executed at cellular replication forks and has crucial implications for the mechanism of MMR, early tumorigenesis, cancer prevention and chemotherapeutics.

Available for download on Tuesday, September 17, 2019

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