Mcm10 promotes rapid isomerization of CMG-DNA for replisome bypass of lagging strand DNA blocks

Abstract

Replicative helicases in all cell types are hexameric rings that unwind DNA by steric exclusion in which the helicase encircles the tracking strand only and excludes the other strand from the ring. This mode of translocation allows helicases to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG helicase partially encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and greatly stimulates its helicase activity in vitro. Most significantly, Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking DNA strand. We demonstrate that bypass occurs without displacement of the blocks and therefore Mcm10 must isomerize the CMG-DNA complex to achieve the bypass function.

The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism

“…In addition, recent studies have indicated that Pol δ sometimes extend the first leading strand primer at an origin before being taken over by Pol ϵ . In vitro assays have identified the minimal protein requirements for both leading and lagging strand synthesis, and for replication fork rates on chromatized DNA consistent with in vivo observations …”

“…Notably, ZF4 binds the future lagging strand in dsDNA, and therefore both strands of the duplex are engaged by CMG, and the unwinding point is at the top of the N‐tier, but just beneath the protruding ZFs, which likely explains inhibition of S.c. CMG by a lagging strand dual SA block . Interestingly, S.c. Mcm10 binds to CMG and enables S.c. CMG to rapidly traverse the dual SA lagging strand block by steric exclusion . The exact mechanism by which Mcm10 promotes this bypass is not yet understood.…”

Replisome‐Cohesin Interfacing: A Molecular Perspective

“…Enigmatic is also the function of the Mcm10 protein which has been involved in both replisome assembly and progression . Mcm10 associates to Mcm2‐7 and Cdc45 and has been proposed to stimulate CMG function and stabilize Pol α …”

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

“…In particular, studies have explored the consequence of placing obstacles on either the translocating or non‐translocating strand using origin replication assays conducted with λ‐phage DNA (Figure F) . Direct encounters between single helicases and steric blocks revealed that both X. laevis and S. cerevisiae CMGs are only affected by a biotin‐streptavidin block on the translocating strand and not the non‐translocating strand . A recent study demonstrates that Mcm10 is required to bypass blocks on the non‐translocating strand either by partial N‐tier ring opening or a dynamic transition between different helicase unwinding modes.…”

“…Direct encounters between single helicases and steric blocks revealed that both X. laevis and S. cerevisiae CMGs are only affected by a biotin‐streptavidin block on the translocating strand and not the non‐translocating strand . A recent study demonstrates that Mcm10 is required to bypass blocks on the non‐translocating strand either by partial N‐tier ring opening or a dynamic transition between different helicase unwinding modes. In addition to a classic steric exclusion mechanism, an alternative proposal involves dsDNA entering the N‐tier and unwinding in the central channel with strand extrusion occurring out the same N‐tier entry .…”