Structural biology of the C-terminal domain of eukaryotic replication factor Mcm10

Abstract

Eukaryotic DNA replication is tightly regulated during the initiation phase to ensure that the genome is copied only once and at the proper time during each cell cycle. During replication initiation, over twenty different proteins are recruited to each origin of replication to denature the DNA duplex and assemble a functional replication fork. Of these, Mcm10 is a DNA binding protein that is recruited to origins in early S-phase and is required for the activation of Mcm2-7, the replicative DNA helicase. Importantly, Mcm10 is necessary for subsequent loading of downstream replication proteins, including DNA polymerase α-primase (pol α), onto chromatin. Mcm10 interacts with single- and double-stranded DNA, pol α, as well as other proteins involved in DNA synthesis. Despite its importance in both replication fork assembly and progression, the precise role of Mcm10 remains undefined. In order to better understand the importance of the molecular interactions of vertebrate Mcm10, we have carried out a structure-function study of the protein from Xenopus laevis (XMcm10), which shares a high sequence homology with the human ortholog. XMcm10 contains three structured regions: a putative oligomerization domain at the N-terminus (NTD) and two independent DNA and pol α binding regions located in the internal (ID) and C-terminal (CTD) domains of the protein. We present a biochemical characterization of the individual domains in Chapter 2, followed by the three-dimensional solution NMR structure and DNA binding activity of the CTD in Chapter 3. The results reveal how the CTD zinc cluster binds DNA and suggests a putative role for this motif in protein-protein interactions with other replisome components. In addition, we show using XMcm10 constructs spanning the two DNA binding domains that the region between is flexible in solution, and that this linker is necessary for optimal DNA binding by XMcm10. Finally, preliminary structural evidence for how the individual ID and CTD modules coordinate DNA binding in the context of the full-length protein is presented in Chapter 4. This modular DNA binding strategy is discussed in terms of Mcm10’s role in during replication initiation and elongation.