| dc.description.abstract | Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase α (pol α), with chromatin. However, the significance of these interactions, and the specific role of Mcm10 during replication initiation is unclear. To address this gap in knowledge, we have begun a structure/function analysis of Xenopus laevis Mcm10 (xMcm10). Chapter 2 describes Mcm10’s domain structure which is composed of three structural and functional regions. The amino-terminal domain (NTD) forms a dimerization motif, while the internal (ID) and carboxy-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss)DNA and the catalytic p180 subunit of pol α. Chapter 3 describes the crystal structure determination of xMcm10-ID as well as NMR spectroscopy to map the binary interfaces between xMcm10-ID and ssDNA. In chapter 4, the mechanism by which Mcm10-ID interacts with pol α is investigated using X-ray crystallography, NMR spectroscopy, isothermal titration calorimetry, and fluorescence anisotropy. In addition, the manner in which the ID and CTD operate together to interact with DNA and pol α are investigated. Collectively, the results presented here provide the first mechanistic insight into how Mcm10 might use a hand-off mechanism to load and stabilize pol α within the replication fork. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and pol α, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome. | |
