Phosphorylation of Mcm2 modulates Mcm2–7 activity and affects the cell’s response to DNA damage

Abstract

The S-phase kinase, DDK controls DNA replication through phosphorylation of the replicative helicase, Mcm2–7. We show that phosphorylation of Mcm2 at S164 and S170 is not essential for viability. However, the relevance of Mcm2 phosphorylation is demonstrated by the sensitivity of a strain containing alanine at these positions (mcm2AA) to methyl methanesulfonate (MMS) and caffeine. Consistent with a role for Mcm2 phosphorylation in response to DNA damage, the mcm2AA strain accumulates more RPA foci than wild type. An allele with the phosphomimetic mutations S164E and S170E (mcm2EE) suppresses the MMS and caffeine sensitivity caused by deficiencies in DDK function. In vitro, phosphorylation of Mcm2 or Mcm2EE reduces the helicase activity of Mcm2–7 while increasing DNA binding. The reduced helicase activity likely results from the increased DNA binding since relaxing DNA binding with salt restores helicase activity. The finding that the ATP site mutant mcm2K549R has higher DNA binding and less ATPase than mcm2EE, but like mcm2AA results in drug sensitivity, supports a model whereby a specific range of Mcm2–7 activity is required in response to MMS and caffeine. We propose that phosphorylation of Mcm2 fine-tunes the activity of Mcm2–7, which in turn modulates DNA replication in response to DNA damage.

The Dbf4-Cdc7 Kinase Promotes Mcm2-7 Ring Opening to Allow for Single-stranded DNA Extrusion and Helicase Assembly

“…suppresser mutations (26,27). Thus, these previous, native promoter experiments should be interpreted with caution.…”

“…Dbf4-Cdc7 phosphorylates Mcm2 in vitro (24,25), but the physiologic role of Dbf4-Cdc7 phosphorylation of Mcm2 is unclear. It has been shown that in vitro, Dbf4-Cdc7 phosphorylates Mcm2 at serines 164 and 170 (25,26). When the gene for mcm2-S164A-S170A (mcm2-2A) is expressed from its endogenous promoter on a plasmid and the cells harboring this plasmid are subjected to a plasmid shuffle assay, no growth defect is observed (26).…”

“…It has been shown that in vitro, Dbf4-Cdc7 phosphorylates Mcm2 at serines 164 and 170 (25,26). When the gene for mcm2-S164A-S170A (mcm2-2A) is expressed from its endogenous promoter on a plasmid and the cells harboring this plasmid are subjected to a plasmid shuffle assay, no growth defect is observed (26). However, in the presence of the replication stress agent methyl methanesulfonate (MMS), a growth defect is observed (26,27).…”

“…When the gene for mcm2-S164A-S170A (mcm2-2A) is expressed from its endogenous promoter on a plasmid and the cells harboring this plasmid are subjected to a plasmid shuffle assay, no growth defect is observed (26). However, in the presence of the replication stress agent methyl methanesulfonate (MMS), a growth defect is observed (26,27). These data led the authors to conclude that Dbf4-Cdc7 is not required for cell growth under normal conditions, but that Dbf4-Cdc7 phosphorylation of Mcm2 may be important during replication stress (26,27).…”

“…However, in the presence of the replication stress agent methyl methanesulfonate (MMS), a growth defect is observed (26,27). These data led the authors to conclude that Dbf4-Cdc7 is not required for cell growth under normal conditions, but that Dbf4-Cdc7 phosphorylation of Mcm2 may be important during replication stress (26,27).…”