Document Type

Dissertation - Open Access

Award Date

2022

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Jaime Lopez-Mosqueda

Abstract

DNA-protein crosslinks (DPCs) form when proteins covalently attach to DNA. It is estimated that hundreds of DPCs form in our cells each day. Because these lesions are cytotoxic, mammalian cells have evolved multiple intricate repair systems to remove DPCs and restore genome integrity. One mechanism mammalian cells rely on for DPC repair is direct proteolysis by the metalloprotease, SPRTN. While it is known that SPRTN degrades a heterogenous array of DPCs, including both naturally occurring and chemically induced, how SPRTN selects DPCs amongst chromatin-associated proteins remains unknown. Our main objective is to determine how DPC repair is regulated. In Chapter 2, we describe our findings that poly (ADP-ribose) polymerase 1 (PARP1) plays a critical role in DPC repair by regulating SPRTN activity towards DPCs. We identified a previously unrecognized Nudix homology domain (NHD) in SPRTN that functions as a direct poly- ADP-ribose interacting region. Interaction with poly-ADP-ribose is required for SPRTNdependent DPC repair. To further demonstrate PARP1 as a key regulator of DPC repair, we found that Topoisomerase 1 covalent complexes (Top1cc) accumulate in cells with compromised PARP1 activity and in cells harboring a SPRTN NHD mutant allele. Likewise, Caenorhabditis elegans expressing the SPRTN NHD mutant as their sole SPRTN source phenocopy SPRTN null nematodes in that they are sensitive to DNA-protein crosslinking agents. Our data implicates PARP1 as an immediate sensor for DPCs and shows that ADP-ribosylation is the biochemical signal that marks DPCs for SPRTNdependent proteolysis. In Chapter 3, we describe methodology we established to quantify DPC repair in C. elegans. Previously, there were no assays adapted for direct analysis of DPC repair in C. elegans. We modified the RADAR assay (rapid approach to DNA adduct recovery) and potassium chloride (KCl)/sodium dodecyl sulfate (SDS) protein precipitation methodology to detect DPCs from C. elegans extracts. These methods are beneficial tools that extend our ability to analyze DPC repair in a new system, C. elegans. Lastly, we conclude with future directions in Chapter 4. DPCs are not only marked with poly-ADP-ribose (PAR); they can also be SUMOylated and/or ubiquitinated to signal for proteasome-mediated degradation. We next aim to understand how the cell coordinates multiple DPC repair pathways and how it decides which DPC repair pathway to employ. We will investigate if PARP1-mediated ADP-ribosylation regulates DPC SUMOylation and ubiquitination. These findings will make strides toward grasping the molecular mechanisms of DPC repair.

Number of Pages

113

Publisher

South Dakota State University

Available for download on Monday, May 15, 2023

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Rights Statement

In Copyright