Author ORCID Identifier

https://orcid.org/0000-0001-7329-9996

Date Available

11-15-2023

Year of Publication

2023

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Toxicology and Cancer Biology

First Advisor

Dr. Eva M. Goellner

Second Advisor

Dr. Isabel Mellon

Abstract

DNA mismatch repair (MMR) is the DNA repair mechanism that repairs base-base mispairs and small insertions and deletions remaining after replication. MMR is also required for apoptosis after certain types of exogenous DNA damage that result in damage-associated mispairs. The basic MMR mechanism is well understood; however, proteins associated with MMR continue to be identified. The roles of these interacting proteins in MMR are largely unknown. We have identified the yeast protein Rad5 as a novel interactor of the critical MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair. However, to date, Rad5 has no known role in MMR despite interacting with both MMR factors. We show that the deletion of yeast RAD5 does not have the mutation rate or mutation spectrum associated with defective canonical MMR. Rad5’s interactions with MMR are conserved throughout evolution and split between its human homologs, HLTF and SHPRH.

Human MSH2 interacts with HLTF regardless of damage, whereas human MLH1 interacts with SHPRH in an MMR-specific damage-dependent manner. Loss of HLTF or SHPRH, alone or in tandem, does not affect canonical MMR. SHPRH knockdown or knockout induces a moderate resistance to MMR-mediated apoptosis; however, loss of HLTF does not affect MMR-mediated apoptosis. We recently confirmed that our HLTF and SHPRH knockout cells affect survival after exposure to DNA-damaging agents that are substrates for post-replicative repair. Loss of MSH2, but not MLH1, also confers a resistance to apoptosis when treated with DNA damage related to post-replicative repair.

This study defines a novel accessory factor that binds with MMR proteins and is conserved from yeast to humans. This study also provides a deeper understanding of how MMR accessory factors may provide a mechanistic distinction between canonical and non-canonical MMR and how MMR influences post-replicative repair pathways. Understanding the interplay between MMR and other repair pathways is essential for cancer development and treatment implications.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2023.422

Funding Information

  • This study was supported by the National Institutes of Health (R00ES026653) from 2020-2022
  • This study was supported by the Markey Foundation Markey Women Strong Distinguished Researcher award in 2020
  • This study was supported by the Phi Beta Psi Cancer Research Grant from 2020-2022
  • This study was supported by the UK-CARES Grant P30 ES026529 Career Development Award from 2020-2022

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