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

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

Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model

“…It is unclear if the C-tier motor ring is able to operate in reverse to pump the DNA inwards. In yet another model, Mcm10 plays a major role in origin melting by binding to and stabilizing the melted and extruded lagging strand ( 42 ). Determining the structures of the activation intermediates of the double hexamer would be a key to clarify the important question in chromosome replication.…”

“…Initially DDK-dependent phosphorylation of Mcm2-7 promotes the recruitment of Sld3 and Cdc45 ( 26 – 34 ) before CDK-dependent phosphorylation of Sld2 and Sld3 ( 35 , 36 ) facilitates the binding of a preloading complex consisting of polymerase ε, GINS, Sld2, and Dpb11 ( 37 ). MCM10 binding is required to activate the CMG helicase for the production of ssDNA and splitting of the Mcm2-7 double hexamer ( 38 – 42 ). The process of helicase activation transforms the Mcm2-7 double hexamer that encircles dsDNA into two Mcm2-7 hexamers that have ssDNA in the central channel, but the mechanistic basis of this transition is not known ( 3 , 43 – 45 ).…”

Noise in the Machine: Alternative Pathway Sampling is the Rule During DNA Replication

“…[95,97,101,102] Second, Mcm10 has a versatile role in origin melting, CMG assembly/stimulation, and Pol α recruitment to CMG. [103] Finally, Csm3/Tof1/Mrc1 may link helicase to polymerase activity [94,104,105] and have recently been shown to stimulate replication to in vivo rates both in bulk and single-molecule assays. [106,107] Taken together, these observations highlight the numerous interactions between CMG and other replisome components that can serve to modulate replication fork progression.…”

“…First, Ctf4, a homotrimeric complex, has been shown to couple CMG and Pol α . Second, Mcm10 has a versatile role in origin melting, CMG assembly/stimulation, and Pol α recruitment to CMG . Finally, Csm3/Tof1/Mrc1 may link helicase to polymerase activity and have recently been shown to stimulate replication to in vivo rates both in bulk and single‐molecule assays .…”

Congenital Diseases of DNA Replication: Clinical Phenotypes and Molecular Mechanisms

“…What is known is that CDC45 and the GINS complex bind across the MCM2/5 gate and may be needed to facilitate ssDNA extrusion or prevent leading strand template escape [ 34 , 35 , 36 ]. MCM10 is required for significant ssDNA generation and subsequent replication protein A (RPA) binding ( Figure 1 ) [ 37 , 38 , 39 ]. MCM10′s role in generating ssDNA during origin firing may be to enable the head-to-head oriented CMG complexes to separate and slide past each other to initiate unwinding [ 40 ].…”