Mcm10-ID is the highly conserved region of the protein that binds both DNA and pol α. The crystal structure of Xenopus Mcm10-ID reveals a unique arrangement of OB-fold and zinc finger motifs. NMR chemical shift perturbation and mutational analysis show that DNA spans both the hydrophobic β barrel of the OB-fold and the electrostatic, extended loop of the zinc finger. This model is further substantiated by the finding that substitutions of conserved key residues within the OB-fold and zinc finger in scMcm10 decrease cell viability in the face of replication stress.
At this time we are unable to reconcile the high-resolution structure of the core domain of Mcm10 with the proposed hexameric structure of the full-length human protein (Okorokov et al., 2007
). Efforts to dock our high-resolution crystal structure into the large volume within the hexameric EM reconstruction did not result in a clear solution. Moreover, the novel OB-fold/zinc finger configuration in Mcm10-ID bears no structural or functional resemblance to the OB-fold/zinc finger domain in the ring-shaped archaeal MCM helicase () (Fletcher et al., 2003
). In addition, burying the ID within a hexameric assembly would likely occlude one or more sites of DNA or protein interactions described below. Nevertheless, the Mcm10 structures serve as an important launching point for further work to investigate the function of this essential eukaryotic replication factor.
The unique DNA binding platform observed here raises interesting questions regarding Mcm10 association with chromatin. Given that Mcm10 possesses two DNA binding domains and can bind to both ss- and dsDNA (Robertson et al., 2008
), it is likely that the protein is anchored to DNA throughout replisome assembly. Interestingly, NMR titration of Mcm10-ID with dsDNA resulted in chemical shift perturbations of largely the same residues as for ssDNA, strongly implying that the binding sites for ssDNA and dsDNA are similar. The binding of both ssDNA and dsDNA to the same site is unexpected given the existence of classic ssDNA and dsDNA binding motifs. Further analysis is required to understand the molecular basis for the complex nature of the DNA binding to Mcm10-ID and the results of such studies will be reported in due course. Nevertheless, the lack of specificity for a particular DNA structure and a common ss/ds-DNA binding site within the ID raises the possibility that Mcm10 acts as a molecular scaffold to stabilize the replisome on DNA. Indeed, the effect of DNA binding residues on HU sensitivity in yeast suggests that Mcm10’s DNA binding function is critical for fork integrity during DNA synthesis.
The structure of Mcm10-ID enables the location of residues that effect DNA replication and cell viability (Supplementary Figure S5
). The cdc23-1E2
Cys239Tyr (Grallert and Nurse, 1997
) and cdc23-M30
Leu287Pro (Liang and Forsburg, 2001
) mutations map to xMcm10 Leu323 and Leu369, respectively, which point into the core of the OB-fold β-barrel and are thus likely to disrupt the protein fold. Similarly, cdc23-M36
Asp232Gly (Nasmyth and Nurse, 1981
) relates to an invariant aspartate (xMcm10 Asp313) on the interior of the L23 loop, and thus likely alters the conformation of L23. This loop might mediate Mcm10-protein interactions given its surface exposed location immediately outside of the DNA binding region. Similarly, cdc23-M36
Val265Ile and mcm10-1
Pro269Leu mutations (Maine et al., 1984
; Nasmyth and Nurse, 1981
) map to solvent exposed positions on L45 and thus likely mediate intermolecular interactions. These residues are adjacent to the putative pol α binding surface, and extensive NMR chemical shift perturbation was observed in the L45 loop upon addition of DNA ().
The relative positions of residues that mediate protein-protein and protein-DNA interactions provide further insight into Mcm10’s role at the replication fork. Of Mcm10’s protein binding partners, PNCA and pol α are the most extensively characterized. The putative PCNA interacting protein (PIP) box predicted from the scMcm10 sequence (Ricke and Bielinsky, 2006
) coincides with the OB-fold β3 strand (Supplementary Figure S5
). scMcm10 Tyr245 was found to be important for an interaction between diubiquinated scMcm10 and PCNA (Das-Bradoo et al., 2006
). This residue (xMcm10 Phe324) is located on the concave, DNA-binding face of β3 () and had a modest effect on DNA binding. We therefore speculate that it might contribute to DNA binding in unmodified Mcm10, but alters its interaction with DNA upon Mcm10 ubiquitination, which has been suggested to trigger a conformational change (Das-Bradoo et al., 2006
). This conformational change likely affects the β3 strand within the OB-fold because extensive mutational analysis suggests that Mcm10 interacts directly with PCNA via its PIP box motif (Das-Bradoo et al., 2006
). Further work to elucidate the site of ubiquitination and its structural consequences will be required to understand the mechanistic basis for how Mcm10 modulates its interactions with DNA and PCNA.
In S. cerevisiae
, the primary interaction site for pol α is confined to a hydrophobic stretch, the heat-shock protein (Hsp)10-like motif (Ricke and Bielinsky, 2006
), which lies adjacent to Mcm10’s PIP box (Das-Bradoo et al., 2006
) within Mcm10-ID. scMcm10 Asn268 (xMcm10 Asn346) is important for Mcm10 stabilization of pol α and maps to the C-terminal end of the β4 strand (Supplementary Figure S5
). We have previously shown that Mcm10-ID binds to the N-terminus of the catalytic p180 subunit of pol α in vitro
(Robertson et al., 2008
). The fact that Asn346 is surface exposed is consistent with a role for this residue in binding pol α. In addition, Asn346 is clearly located outside of the DNA binding interface. This raises the possibility that Mcm10-ID can bind DNA and pol α simultaneously and is consistent with the proposal that Mcm10 acts to recruit pol α to the origin (Ricke and Bielinsky, 2004
). Furthermore, evidence is mounting to suggest that Mcm10 likely associates with pol α during elongation (Chattopadhyay and Bielinsky, 2007
; Pacek et al., 2006
; Ricke and Bielinsky, 2004
; Yang et al., 2005
). Structures of Mcm10 in complex with its protein binding partners will be critical to understand the physical basis for function of this modular, multi-functional protein.