Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model

Progression of DNA replication depends on the ability of the replisome complex to overcome nucleoprotein barriers. During eukaryotic replication, the CMG helicase translocates along the leading-strand template and unwinds the DNA double helix. While proteins bound to the leading-strand template efficiently block the helicase, the impact of lagging-strand protein obstacles on helicase translocation and replisome progression remains controversial. Here, we show that CMG and replisome progressions are impaired when proteins crosslinked to the lagging-strand template enhance the stability of duplex DNA. In contrast, proteins that exclusively interact with the lagging-strand template influence neither the translocation of isolated CMG nor replisome progression in Xenopus egg extracts. Our data imply that CMG completely excludes the lagging-strand template from the helicase central channel while unwinding DNA at the replication fork, which clarifies how two CMG helicases could freely cross one another during replication initiation and termination. [Display omitted] •The CMG helicase can bypass a lagging-strand DNA-protein crosslink without stalling•A methyltransferase on the lagging strand stalls CMG by stabilizing duplex DNA•Single-molecule detection of CMG pausing at a lagging-strand methyltransferase•Replisome progression matches the dynamics of purified CMG at identical roadblocks During DNA replication, the CMG complex unwinds the double helix and must overcome protein barriers on DNA. Kose et al. show that proteins that interact exclusively with the lagging-strand template are bypassed by the helicase without stalling, implying that the lagging-strand template is completely excluded from CMG during unwinding.