Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cellular Biology
Washington, M. Todd
First Committee Member
Second Committee Member
Third Committee Member
Fourth Committee Member
Proliferating cell nuclear antigen (PCNA) is a versatile protein involved in all pathways of DNA metabolism. It is best known as a processivity factor for classical polymerases, which synthesize DNA on non-damaged templates during DNA replication (ex: pol δ). Non-classical polymerases, on the other hand, are those that synthesize DNA on damaged templates (ex: pol η). PCNA also functions in repair, recombination, and most other DNA-dependent cellular processes. A number of separation of function mutant PCNA proteins have been identified, suggesting that PCNA could be a valuable target to manipulate DNA metabolism. This thesis focuses on the study of PCNA mutant proteins that affect translesion synthesis (TLS) and mismatch repair (MMR).
During TLS, the process by which DNA polymerases replicate through DNA lesions, PCNA recruits and stabilizes polymerases at the replication fork. TLS requires the monoubiquitylation of PCNA, and PCNA and ubiquitin-modified PCNA (Ub-PCNA) stimulate TLS by classical and non-classical polymerases. Two mutant forms of yeast PCNA, one with an E113G substitution and one with a G178S substitution, support normal cell growth but inhibit TLS. To better understand the role of PCNA in TLS, I re-examined the structures of both mutant PCNA proteins and identified substantial disruptions of the subunit interface that forms the PCNA trimer. This resulted in reduced trimer stability in the mutant proteins. The mutant forms of PCNA and Ub-PCNA do not stimulate TLS of an abasic site by either classical pol δ or non-classical pol η. Normal replication by pol η was also impacted, but normal replication by pol δ was much less affected. These findings support a model in which reduced trimer stability causes these mutant PCNA proteins to occasionally undergo conformational changes that compromise their ability to stimulate TLS by both classical and non-classical polymerases.
During MMR, PCNA recruits and coordinates proteins involved in the mismatch recognition, excision, and resynthesis steps. Previously, two mutant forms of PCNA were identified that cause defects in MMR with little if any other defects. These are the C22Y and C81R mutant PCNA proteins. In order to understand the structural and mechanistic basis by which these two substitutions in PCNA proteins block MMR, we solved the X-ray crystal structures of both mutant proteins and carried out further biochemical studies. I found that these amino acid substitutions lead to distinct structural changes in PCNA. The C22Y substitution alters the positions of the α-helices lining the central hole of the PCNA ring, whereas the C81R substitution creates a distortion in the β-sheet at the PCNA subunit interface. I conclude that the structural integrity of the α-helices lining the central hole and the β-sheet at the subunit interface are both necessary to form productive complexes with MutSα and mismatch-containing DNA.
As described above, my studies focused on four amino acid substitutions in PCNA that disrupt TLS and MMR: the E113G and G178S substitutions cause defects in TLS while the C22Y and C81R substitutions cause defects in MMR. The structures of these mutant PCNA proteins revealed that three of the four substitutions caused disruptions near the subunit interface of PCNA. To further examine the importance of this region, we generated random mutations of the PCNA subunit interface and performed in vivo genetics assays and in vitro biochemical assays to examine their effects on TLS and MMR. We determined that the subunit interface of PCNA is very dynamic and that small changes at this interface can cause drastically different effects on TLS and MMR. Moreover, we suggest that the integrity of the subunit interface as well as the nearby β-strands in domain A are crucial for proper PCNA function in vivo and in vitro.
xiv, 235 pages
Includes bibliographical references (pages 213-235).
Copyright 2013 Lynne Margaret Dieckman
Dieckman, Lynne Margaret. "Studies of Proliferating Cell Nuclear Antigen Mutant Proteins Defective in Translesion Synthesis and Mismatch Repair." PhD (Doctor of Philosophy) thesis, University of Iowa, 2013.