Role of WD domain-containing proteins in cell cycle progression

Cell cycle;Deoxyribonucleic acid (DNA) replication;Chromatin organization

Wang, Yating

关键词: Cell cycle;    Deoxyribonucleic acid (DNA) replication;    Chromatin organization;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/98255/WANG-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

In the eukaryotic proteome, WD domain is one of the most abundant protein motifs. The structure of WD domain allows it to interact with diverse protein partners and proteins containing WD domain have been shown to function in various cellular processes. In this thesis, I describe how two WD domain-containing proteins, LRWD1/ORCA and RFWD3, regulate different aspects of cell cycle progression.In eukaryotic cells, the duplication of the genome starts from the loading of a six-subunit complex, Origin Recognition Complex (ORC), onto replication origins on the chromosome. The loading of ORC triggers the assembly of the pre-replicative complex (pre-RC) at origins, which licensed the origins prior to entering S phase. Unlike yeast, replication origins in higher eukaryotes do not have consensus sequence and metazoan ORC does not bind to DNA in a specific manner. Thus, a long-standing question in the field has been, how are origins specified and how are ORCs recruited to origins? Studies suggest that ORC loading may be facilitate by ORC-associated proteins. LRWD1/ORCA is an ORC-associated protein that stabilizes ORC on chromatin. We have previously characterized ORCA’s role in replication initiation and heterochromatin organization. In Chapter 2, I describe my work to map the binding sites of ORCA genome-wide by ChIP- seq method at specific time points of G1. ORCA associates with chromatin in a dynamic manner and ORCA binding regions are enriched for heterochromatic marks, including H3K9me3 and methylated-CpGs. ORCA co-localizes with a subset of origins that are enriched for repressive marks, consistent with the fact that these origins replicate late during S phase. Further, ORCA directly associates with the repressive marks and interacts with the enzymes that catalyze these marks. ORCA regulates the level of H3K9me3 and methylated-CpG at its binding sites, suggesting a role of ORCA in maintaining the repressive chromatin marks at its binding sites. Importantly, repressive marks are required for ORCA’s association on chromatin. I propose that ORCA localizes to a subset of origins by association with repressive marks and recruits the enzymes to maintain the repressive chromatin environment.In Chapter 3, I focus on another WD domain containing protein, RFWD3, and describe how RFWD3 regulates cell cycle progression. Unlike previous studies that described how RFWD3 regulates DNA damage response, I focus on how RFWD3 regulate unperturbed cell cycle progression. I show that RFWD3 is required for proper cell cycle progression. Depletion of RFWD3 causes S phase defects including slower replication fork progression and prolonged S phase. There is also increased loading of replication fork proteins on S phase chromatin in the absence of RFWD3, potentially due to stalled replication fork. RFWD3 associates with the replication fork component, PCNA, via a PIP box motif. I purpose that RFWD3 localizes to replication fork by interacting with PCNA and regulate proper replication fork progression.In Chapter 4, I summarize my findings on the regulation of DNA replication, chromatin organization and S phase progression. I also discussed interesting areas of research that my work has opened up.