| dc.description.abstract | Faithful DNA replication during each cell division cycle is carried out by multiprotein complexes acting at each replication fork. In this complex, DNA unwinding and synthesis activities, driven by the Mcm2-7 helicase and DNA polymerases α, delta and ε, are coordinated by non-enzymatic replication factors. One such protein, Mcm10 (mini-chromosome maintenance protein 10) interacts with single- and double-stranded DNA, pol α, and Mcm2-7. Mcm10 loads onto chromatin at the onset of S phase and is required for subsequent recruitment of downstream proteins, and is thus critical for the replisome assembly during replication initiation. Mcm10 has been reported to form homodimeric, trimeric, and hexameric assemblies. Vertebrate Mcm10 protein consists of an N-terminal oligomerization domain (NTD), followed by internal (ID) and C-terminal domains (CTD). ID and CTD each binds DNA and pol α. The NTD, containing a coiled-coil motif, was shown by analytical ultracentrifugation (AUC) to form a dimer, and deletion of this domain from the full-length protein results in a monomeric Mcm10 form. This dissertation describes structure-function analyses of the coiled-coiled (CC) region. AUC showed that the CC exists as dimer and trimer in solution, with trimeric assemblies stabilized by low pH. Crystal structure at 2.4 Å resolution at acidic pH showed the coiled-coil region as a 3-helix bundle. Mutation of aliphatic residues at the coiled-coil interface disrupted oligomerization of the CC and NTD observed by AUC. The same mutants disrupted FL Mcm10 oligomerization in vivo detected by yeast-2-hybrid. These studies more clearly define the role of the NTD in Mcm10 oligomerization and provide insights into the biological function of Mcm10 in DNA replication. | |
