Date of Award

2019

Embargo Period

8-1-2024

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

David T. Long

Second Advisor

J. Alan Diehl

Third Advisor

Stephen P. Ethier

Fourth Advisor

Philip H. Howe

Fifth Advisor

Elizabeth S. Yeh

Abstract

p97/VCP is a highly abundant, homohexameric AAA+ ATPase that performs a variety of essential cellular functions. Characterized as a ubiquitin-selective chaperone, p97 recognizes proteins conjugated to K48-linked polyubiquitin chains and promotes their removal from chromatin and other protein complexes. Previously, the exact role p97 plays in DNA inter-strand crosslink repair was speculative and undetermined. In 2014, it was shown that p97 is able to promote unloading of the CMG helicase at the end of DNA replication. Similarly, when the CMG helicase collides with an inter-strand crosslink, it too must be evicted to allow repair enzymes to access the lesion. Using Xenopus egg extracts to model DNA inter-strand crosslink repair during replication, we showed that p97 promotes CMG removal from DNA in a BRACA1-dependent manner. Changes in p97 activity are associated with cancer progression and several related neurodegenerative disorders. Although pathogenic p97 mutations cluster in and around p97’s ATPase domains, the majority of mutant proteins display normal or elevated ATPase activity. How pathogenic mutations in p97 disrupt function remains unclear. Using Xenopus egg extracts to model protein extraction from chromatin, we showed that one of the most common p97 mutations (R155C) retains ATPase activity, is recruited to ubiquitylated substrates on chromatin, but cannot promote substrate removal. As a result, p97-R155C acts as a dominant negative, blocking protein extraction, while consuming excess ATP which can hinder high-energy cellular processes. Together, our results shed new insight regarding p97’s chromatin-associated functions with important implications for understanding the etiology and treatment of p97-associated diseases.

Rights

All rights reserved. Copyright is held by the author.

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