Molecular mechanism of the eukaryotic replicative helicase

Abstract

In eukaryotes, the replicative helicase CMG (Cdc45/Mcm2-7/GINS) complex separates the two strands of the double helix during DNA replication. A number of models have been proposed to explain how CMG translocates on DNA during unwinding. To differentiate among different models and to understand the way CMG deals with protein barriers on DNA, we performed DNA unwinding assays using purified CMG and various DNA substrates containing site specific protein adducts. We showed that CMG can unwind a DNA substrate containing a protein barrier on the lagging-strand template, and that unwinding is blocked by a protein adduct on the leading-strand template. We also show that inhibition of lagging strand roadblock bypass only occurs when CMG binding is impaired. Furthermore, we found that while a DNA-protein crosslink that exclusively interacts with the lagging-strand template does not affect translocation kinetics of CMG, methyltransferase enzyme crosslinked to the lagging-strand template stabilizes duplex DNA and leads to a delay in unwinding. Using electron microscopy, we visualized CMG bound to a fork DNA substrate and found that the helicase is oriented with the N-terminal domains of Mcm2-7 facing the fork junction. Importantly, high-resolution cryo-EM structure of Drosophila melanogaster CMG (DmCMG) bound to fork DNA substrate revealed that DmCMG partially encircles duplex DNA at the fork junction. Strikingly, capture of the excluded strand with a complementary oligonucleotide or RPA stimulates DNA unwinding, implying that engagement of CMG with duplex DNA at the fork junction impairs its helicase activity. We propose that duplex engagement at the fork junction does not reflect translocating state of CMG, rather paused state of CMG. Together, our data indicate that two Mcm2-7 hexamers, encircling duplex DNA at origins, are remodeled to completely exclude one strand from the ring channel and engagement with the duplex DNA after helicase activation impairs translocation.