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Title:Role of replication proteins in the maintenance of genome integrity
Author(s):Lin, Yo-Chuen
Director of Research:Prasanth, Supriya G
Doctoral Committee Chair(s):Prasanth, Supriya G
Doctoral Committee Member(s):Belmont, Andrew S; Chen, Jie; Nair, Satish K
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):DNA replication
DNA repair
DNA damage response
Abstract:Precise duplication of our genome is critical for cell survival and human health. Meanwhile, appropriate cellular response to genotoxic stress during DNA replication prevents genome instability and ensures the fidelity of replication. In this thesis, I first review and discuss the current understanding of how DNA replication is regulated from G1 origin licensing to S phase replication elongation, as well as the coordination between replication and DNA repair/DNA damage response. I then describe in Chapters 2 and 3 the studies of two important replication proteins, Orc6 and RFWD3, in the maintenance of genome integrity. In Chapter 2, I investigate the functional significance of the smallest origin recognition complex subunit, Orc6. In eukaryotes, the Origin Recognition Complex (ORC) is required for the initiation of DNA replication. As part of the ORC, it has been reported that Orc6 is essential for pre-replication complex (pre-RC) assembly and cell viability in yeast and Drosophila. However, the role of human Orc6 in replication remains unclear. I characterize human Orc6 function for G1 origin licensing versus S phase progression. I demonstrate that in human cells, Orc6 is dispensable for G1 licensing, opposing to its suggested role for MCM loading observed in other species. Instead, I identify an unexpected role for human Orc6, which is to promote S-phase progression post pre-RC assembly and regulate DNA damage response. Moreover, Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S-phase, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutSα and enhances the association of MutLα to MutSα, thus enabling efficient mismatch repair. This study here reveals that Orc6 plays a fundamental role in genome surveillance during S-phase. In Chapter 3, I focus on RFWD3, an E3 ligase known to facilitate homologous recombination by removing RPA and RAD51 from DNA damage sites. The role of RFWD3 in DNA damage conditions has been extensively studied. However, the role of RFWD3 in unperturbed cell cycle progression remains to be solved. Preliminary data from our lab have shown that RFWD3 interacts with Proliferating Cell Nuclear Antigen (PCNA) via its PCNA-interacting (PIP) motif. Further, knockdown of RFWD3 causes slower replication fork progression. Here, I demonstrate that in unperturbed human cells, the interaction with PCNA stabilizes RFWD3 at the replication fork. Cells lacking RFWD3 show a prolonged S-phase and replication stress phenotype with increased frequency of fork stalling. The S-phase defect is rescued by WT-RFWD3, but not by the PIP-mutant, suggesting that the interaction of RFWD3 with PCNA is critical for DNA replication. Finally, I observe reduced ubiquitination of RPA in cells lacking RFWD3, suggesting an inefficient RPA removal during replication progression. Together, I propose that the stabilization of RFWD3 by PCNA at the replication fork enables the polyubiquitination of RPA and its subsequent degradation for proper DNA replication. In Chapter 4, I summarize the significance of my findings of these two critical replication proteins, Orc6 and RFWD3, in regulating genome integrity. I also discuss several interesting questions that have emerged from my research, as well as future directions for their research.
Issue Date:2020-11-20
Type:Thesis
URI:http://hdl.handle.net/2142/109584
Rights Information:Copyright 2020 Yo-Chuen Lin
Date Available in IDEALS:2021-03-05
Date Deposited:2020-12


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