Solution NMR structure of the C-terminal DNA binding domain of Mcm10 reveals a conserved MCM motif.
The eukaryotic DNA replication protein Mcm10 associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Xenopus laevis (X) Mcm10 binds DNA via a highly conserved internal domain (ID) and a C-terminal domain (CTD) that is unique to higher eukaryotes. Although the structural basis of the interactions of the ID with DNA and polymerase alpha is known, little information is available for the CTD. We have identified the minimal DNA binding region of the XMcm10-CTD and determined its three-dimensional structure by solution NMR. The CTD contains a globular domain composed of two zinc binding motifs. NMR chemical shift perturbation and mutational analysis show that ssDNA binds only to the N-terminal (CCCH-type) zinc motif, whose structure is unique to Mcm10. The second (CCCC-type) zinc motif is not involved in DNA binding. However, it is structurally similar to the CCCC zinc ribbon in the N-terminal oligomerization domain of eukaryotic and archaeal MCM helicases. NMR analysis of a construct spanning both the ID and CTD reveals that the two DNA binding domains are structurally independent in solution, supporting a modular architecture for vertebrate Mcm10. Our results provide insight in the action of Mcm10 in the replisome and support a model in which it serves as a central scaffold through coupling of interactions with partner proteins and the DNA.
Robertson
Patrick D
PD
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232, USA.
Chagot
Benjamin
B
Chazin
Walter J
WJ
Eichman
Brandt F
BF
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D064110:Minichromosome Maintenance Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D014982:Xenopus laevis; D015032:Zinc
D004268:DNA-Binding Proteins; C527642:Mcm10 protein, Xenopus; D004247:DNA; D064110:Minichromosome Maintenance Proteins; D015032:Zinc