Document Type

Thesis - Open Access

Award Date

2023

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Jaime Lopez-Mosqueda

Second Advisor

Radhey Kaushik

Abstract

DNA-protein crosslinks (DPCs) are a type of DNA lesion that form when proteins become covalently linked to DNA. It is estimated that replicating cells experience approximately 6,000 DPCs per day per genome during exponential growth (Ruggiano & Ramadan, 2021). If left unrepaired, DPCs can be lethal to cells. For this reason, cells have evolved multiple pathways to repair or bypass DPCs to survive. One such pathway involves SPRTN, a nuclear metalloprotease that plays a key role in the repair of DPCs through direct proteolysis (Lopez-Mosqueda et al., 2016; Vaz et al., 2016). Once SPRTN degrades the bulky protein component of the DPC, additional repair enzymes are able to access the damage site and restore the DNA. We recently demonstrated that poly (ADP-ribose) polymerase 1 (PARP1) modifies DPCs with poly-ADP-ribose (PAR) to signal for SPRTN-dependent degradation. PARP1 is an abundant DNA repair enzyme that catalyzes ADP-ribosylation (ADPr) to regulate cellular processes in addition to DNA repair, including chromatin remodeling, telomere maintenance, and gene transcription (Saha et al., 2021). While it is known that SPRTN displays promiscuous proteolytic activity in vitro, and it is likely that SPRTN activity is tightly regulated in cells to prevent accidental degradation of essential enzymes, how SPRTN activity is regulated is not completely understood. Previous efforts have shown that SPRTN can be post-translationally modified with ubiquitin and acetyl groups (E. J. Davis et al., 2012; Huang et al., 2020; Perry et al., 2021; Stingele et al., 2016). Mono-ubiquitylation of SPRTN is thought to block its access to chromatin, while deubiquitination of SPRTN by recently identified deubiquitin hydrolases (DUBs) promote its access to chromatin (Huang et al., 2020; Perry et al., 2021; Zhao et al., 2021). Furthermore, acetylation of SPRTN promotes DPC repair, though the exact mechanisms are unknown. We recently identified that SPRTN is post-translationally modified with poly-ADP-ribose (PAR). This particular post-translational modification on SPRTN has not been previously reported. Given that PAR is negatively charged, we hypothesize that it could function to repel SPRTN from chromatin and therefore negatively regulate SPRTN activity. The role of SPRTN in repairing DPCs on damaged DNA strands is established and confirmed by several laboratories. This work herein demonstrates that SPRTN is PARylated by PARP1 and aims to establish and characterize SPRTN PARylation. In Chapter 2, we serendipitously detected a post-translational modification on SPRTN using an anti-SPRTN antibody developed in-house. We utilize PARG inhibitor (PARGi) and PARP inhibitor (PARPi) to analyze the functional role of PARP1-mediated ADPr in SPRTN-dependent DNA damage repair. Our data suggests that PARP1 and SPRTN work closely together to regulate SPRTN-dependent DPC repair.

Library of Congress Subject Headings

DNA repair.
DNA damage.
ADP-ribosylation.
DNA-protein interactions.
Proteins -- Crosslinking.
DNA replication.

Publisher

South Dakota State University

Download

Share

COinS
 

Rights Statement

In Copyright