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

Nucleic Acids Research
Patricia Perez-ArnaizDaniel L Kaplan

Abstract

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 ...Continue Reading

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Citations

Oct 29, 2017·Proceedings of the National Academy of Sciences of the United States of America·Yasunori NoguchiHuilin Li
Dec 29, 2017·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Matthias J ScherrKarl E Duderstadt
Jan 23, 2021·International Journal of Molecular Sciences·Megan Schmit, Anja-Katrin Bielinsky

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Methods Mentioned

BETA
electron microscopy
PCR
electrophoretic mobility shift
pulldown
immunoprecipitation
co-immunoprecipitation
electrophoretic mobility
electrophoretic mobility gel shift
FACS

Software Mentioned

Image Studio
Diva
FACS

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