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Title:ORCA/LRWD1, a novel player in the initiation of eukaryotic DNA replication
Author(s):Shen, Zhen
Director of Research:Prasanth, Supriya G.
Doctoral Committee Chair(s):Belmont, Andrew S.
Doctoral Committee Member(s):Prasanth, Supriya G.; Freeman, Brian C.; Yau, Peter M.; Cann, Isaac K.
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):ORC-associated (RCA)
origin recognition complex
pre-replicative complex
DNA replication
leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1)
Abstract:Accurate DNA replication is key to maintaining genome stability in all living species. In eukaryotes, the duplication process starts with the formation of the pre-replicative complex (pre-RC) prior to S phase, followed by the transition to the pre-initiation complex (pre-IC) that is required for DNA synthesis. The conserved canonical model of the pre-RC assembly includes an ordered recruitment of ORC, Cdt1, Cdc6, and the helicase MCMs to replication origins, and is strictly governed by replication licensing system. As the complexity increases dramatically from yeast to human cells, additional replication regulators have been identified in higher eukaryotes. In Chapter I, I review how the canonical pre-RC components were identified; what are the novel players; and our current understanding of how the replication licensing dictates proper DNA synthesis. While how ORC is recruited to specific origins has been studied explicitly in lower organisms, it remains unclear in higher eukaryotes. To understand whether additional factors enable the recruitment of ORC to origins and/or specific chromatin sites in human cells, our lab conducted ORC immunoprecipitations followed by mass spectrometric analysis in order to find novel ORC interactors. In Chapter II, I describe the identification of a highly conserved protein in human cells, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA). It interacts with ORC and modulates its chromatin association. ORCA co-localizes with ORC, shows similar cell cycle dynamics, and efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in the loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation of cells in G1 phase. I therefore propose the model where ORCA-mediated association of ORC to chromatin is critical for pre-RC assembly in G1. In addition to ORC, I describe the interaction of ORCA with replication factor Cdt1 and its inhibitor Geminin in Chapter III. I demonstrate the stoichiometry of these associations via single-molecule pull-down experiments that each molecule of ORCA can bind to one molecule of ORC, one molecule of Cdt1, and two molecules of Geminin. Further, ORCA directly interacts with the N-terminus of Orc2, and this association is essential for ORCA stability. ORCA associates with Orc2 throughout the cell cycle, with Cdt1 during mitosis and G1, and with Geminin in post-G1 cells. Interestingly, overexpression of Geminin results in the loss of interaction between ORCA and Cdt1, suggesting that the increased levels of Geminin in post-G1 cells titrate Cdt1 away from ORCA. Based on these data, I propose that the dynamic association of ORCA with pre-RC components modulates the assembly of its interacting partners on chromatin and plays a key role in replication initiation. ORCA exhibits cell cycle-regulated protein levels that peak in G1; and it can only be stabilized when it is in a complex with Orc2. In Chapter IV, I show that ORCA is polyubiquitinated in vivo with elevated ubiquitination observed at the G1/S boundary. ORCA utilizes lysine-48 (K48) ubiquitin linkage suggesting that ORCA ubiquitination mediates its regulated degradation. Ubiquitinated ORCA is re-localized in the form of nuclear aggregates and is predominantly associated with chromatin. I further demonstrate that ORCA ubiquitinates at the WD40 repeat domain, the same region recognized by Orc2. Moreover, Orc2 associates only with the non-ubiquitinated form of ORCA, and Orc2 depletion results in the proteasome-mediated destabilization of ORCA. Based on these results, I propose that Orc2 protects ORCA from degradation by preventing its ubiquitination within the WD domain. In Chapter V, I summarize the significance of my thesis work. With the discovery of a novel component of eukaryotic pre-RC, ORCA, we have better understanding of the eukaryotic DNA replication machinery. I will point out several interesting questions that have emerged from my studies that demand future investigation.
Issue Date:2013-02-03
Rights Information:Copyright 2012 Zhen Shen
Date Available in IDEALS:2013-02-03
Date Deposited:2012-12

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