An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation

作者：Perez Arnaiz Patricia; Kaplan Daniel L

来源：Journal of Molecular Biology, 2016, 428(23): 4608-4625.

DOI：10.1016/j.jmb.2016.10.014

• DNA replication
• yeast
• helicase
• DNA unwinding
• CMG complex

Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bobl bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bobl mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bobl background. Thus, the origin melting and GINS Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bobl background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication.

• 出版日期2016-11-20