An intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly and the recruitment of Pol-α to Mcm2-7.

Mcm10 is an essential eukaryotic factor required for DNA replication. The replication fork helicase is composed of Cdc45, Mcm2-7 and GINS (CMG). DDK is an S-phase-specific kinase required for replication initiation, and the DNA primase-polymerase in eukaryotes is pol α. Mcm10 forms oligomers in vitro, mediated by the coiled-coil domain ...
at the N-terminal region of the protein. We characterized an Mcm10 mutant at the N-terminal Domain (NTD), Mcm10-4A, defective for self-interaction. We found that the Mcm10-4A mutant was defective for stimulating DDK phosphorylation of Mcm2, binding to eighty-nucleotide ssDNA, and recruiting pol α to Mcm2-7 in vitro. Expression of wild-type levels of mcm10-4A resulted in severe growth and DNA replication defects in budding yeast cells, with diminished DDK phosphorylation of Mcm2. We then expressed the mcm10-4A in mcm5-bob1 mutant cells to bypass the defects mediated by diminished stimulation of DDK phosphorylation of Mcm2. Expression of wild-type levels of mcm10-4A in mcm5-bob1 mutant cells resulted in severe growth and DNA replication defects, along with diminished RPA signal at replication origins. We also detected diminished GINS and pol-α recruitment to the Mcm2-7 complex. We conclude that an intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly, and the recruitment of pol α to Mcm2-7.
Mesh Terms:
Animals, Cell Cycle Proteins, DNA Polymerase I, DNA Replication, DNA-Binding Proteins, Humans, Kinetics, Mice, Minichromosome Maintenance Proteins, Mutation, Nuclear Proteins, Phosphorylation, Protein-Serine-Threonine Kinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Xenopus laevis
Nucleic Acids Res.
Date: Jul. 07, 2017
Download Curated Data For This Publication
206329
Switch View:
  • Interactions 27