Origin Recognition Complex (ORC) binding to DNA replication origin is the first step of human DNA replication. However, the mechanisms behind origin of replication firing in higher eukaryotes are poorly understood. SET8 is the methyltransferase responsible for histone H4 lysine 20 monomethylation (H4K20me1). This post-translational modification of H4 histone tail can specifically modulate chromatin structure, which potentially provides a chromatin-dependent mechanism for the recruitment of the human ORC. In this study, we sought to determine how SET8 links chromatin regulation to cell cycle progression in two aspects: investigating novel functions and regulatory mechanisms of SET8, and determining the structures of human Orc6 which is one of the subunit of ORC, and Orc6-DNA com...[ Read more ]

Origin Recognition Complex (ORC) binding to DNA replication origin is the first step of human DNA replication. However, the mechanisms behind origin of replication firing in higher eukaryotes are poorly understood. SET8 is the methyltransferase responsible for histone H4 lysine 20 monomethylation (H4K20me1). This post-translational modification of H4 histone tail can specifically modulate chromatin structure, which potentially provides a chromatin-dependent mechanism for the recruitment of the human ORC. In this study, we sought to determine how SET8 links chromatin regulation to cell cycle progression in two aspects: investigating novel functions and regulatory mechanisms of SET8, and determining the structures of human Orc6 which is one of the subunit of ORC, and Orc6-DNA complex.

In the first part of the thesis, the functional study of SET8 is described. SET8 has two functional domains, including an N-terminal histone binding domain and a C-terminal SET domain. Functions of SET8 are related to DNA replication and transcriptional regulation. In this study, SET8 is found to interact to SUMO E2 and E3 ligases. Sumoylation assay shows that SET8 has three sumoylation sites and SUMO E3 ligase facilitates sumoylation of SET8. The intracellular localization of SET8 is investigated and it is found that SET8 contains a nuclear localization sequence that regulates localization of SET8. In addition, expressions of SET8 in brain and testis were also investigated. The immunofluorescence staining of mouse brain sections displayed that SET8 expresses in neuron but not glia, and it is critical for neuron and mouse survival. The expression pattern of SET8 in testis implies that SET8 plays roles in meiosis. Taken together, this study demonstrates SET8 having novel functions and regulatory mechanism.

In the second part, the structural studies of human Orc6 (hOrc6) and hOrc6-DNA complex are described. The ORC is a protein complex which contains six-subunit (Orc1-6). It plays roles in the initiation of DNA replication in eukaryotic cells. Orc6 is the smallest and the least conserved among all the ORC subunits. In budding yeast, unlike Orc1-5, Orc6 is not required for the origin binding, but for cell proliferation and viability. In contrast, Orc6 is an integral part of ORC in Drosophila and is essential for both DNA binding and replication activity. Currently, the mechanism for diverse functions of Orc6 is not clear. In this study, we determined the structure of core replicative domain of human Orc6 by NMR and confirmed the directly interaction between hOrc6 with DNA. Our results indicate that Orc6 has a two-domain alpha-helical structure. This structure contains two structurally similar repeated helical motifs, and only the domain II of hOrc6 directly binds to DNA in a sequence-independent manner. Compared to the previously published predicted model, our NMR data indicates that more residues of domain II are involved in direct binding and domain I also cooperates this binding.