Single-Molecule Studies of the Eukaryotic Replicative DNA Helicase MCM2-7

Abstract

In eukaryotes, double hexamers of the replicative DNA helicase MCM2-7 are loaded onto double-stranded DNA (dsDNA) at each origin of replication in the G1 phase of the cell cycle. In S phase, numerous accessory factors activate the helicase activity of MCM2-7, leading to unwinding of the DNA template. Despite decades of study, it remains unclear how MCM2-7 unwinds DNA. One model is that upon activation, MCM2-7 goes through a conformational change to encircle single-stranded DNA (ssDNA) whereupon it translocates along one strand while excluding the other (steric exclusion). An alternative model envisions that MCM2-7 translocates along dsDNA. As DNA emerges the rear exit channel of MCM2-7, it is split by a rigid pin that bisects the channel. To distinguish between the two scenarios, we used an experimental system that allows single-molecule visualization of DNA replication in Xenopus egg extracts. We attached a quantum-dot (Qdot) to lambda DNA at a specific location that served as a road block for replication forks. Upon exposure of such Qdot-labeled lambda DNA to extracts in a microfluidic flow cell, a significant fraction of forks bypassed the Qdot when it was located on the lagging strand template but not when it was located on the leading strand template. Our results support the model that MCM2-7 translocates in the 3’ to 5’ direction along ssDNA and unwinds DNA by sterically excluding the opposite strand. Our results suggest that the large number of factors that are required to activate the MCM2-7 complex at the G1/S transition function by remodeling the MCM2-7 complex from a dsDNA binding mode to a ssDNA binding mode.