Date of Award

Fall 10-14-2026

Embargo Period

10-14-2031

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Natalie Saini

Second Advisor

Jessica Hartman

Third Advisor

David Long

Fourth Advisor

Antonis Kourtidis

Fifth Advisor

Ryan Barnes

Abstract

Formaldehyde (FA) and methylglyoxal (MG) are endogenously produced and environmentally abundant nucleophiles capable of inducing base adducts, DNA-DNA crosslinks, and DNA-protein crosslinks.  Aldehydes have been shown to generate damage within regions of ssDNA, but the pathways responsible for responding to this damage has remained unclear.

In chapter two of this dissertation, we explore how translesion synthesis (TLS) and DNA-protein crosslink (DPC) repair coordinate to bypass FA-induced DNA damage and their impact on the mutational spectrum. Using a previously established reporter system in S. cerevisiae, we show that FA preferentially mutagenizes guanine residues within ssDNA, and that the accumulation of these mutations depends on TLS.  In the absence of TLS, we observe large-scale chromosomal instability that is dependent on DPC repair for efficient resolution.

In chapter three of this dissertation, we expand on our findings by showing that FA preferentially mutagenizes endogenously produced ssDNA at dysregulated DNA replication forks. Again, this mutagenesis is dependent on TLS and DPC repair is required for cellular survival following FA exposure. We find that the replication fork protection complex factor Mrc1 plays an integral role in protection against FA-induced mutagenesis and that this function is separate from its checkpoint signaling role.

Finally, chapter four explores molecular mechanisms for MG-induced genotoxicity in human cells. We find that acute MG exposure results in the activation of both ATR and ATM. Interestingly, we find that acute MG exposure results in the depletion of the critical tumor suppressors BRCA1 and BRCA2. This depletion appears to be both at the protein and RNA level as the depletion is rescued by proteasomal inhibition and BRCA1 transcript levels were reduced following MG exposure.  Most notably, we find that BRCA1 and BRCA2 protein levels can be rescued upon co-treatment with the PARP1 inhibitor Olaparib via a currently unknown mechanism.

Overall, this work establishes FA as preferentially targeting ssDNA for damage and TLS-dependent mutagenesis. We also establish novel mechanisms for MG-induced genotoxicity. Since many cancers have elevated aldehyde levels, dual inhibition of aldehyde detoxifying enzymes and specific DNA repair proteins may synergize to form an interesting avenue for future cancer therapies. For these therapies to be successful, we must gain a deeper understanding regarding the genotoxic effects of aldehydes and the DNA repair pathways that respond to these insults.

Rights

Copyright is held by the author. All rights reserved.

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