| Summary: | 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 ...
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