Mcm10 functions to isomerize CMG-DNA for replisome bypass of DNA blocks

1 2 Replicative helicases of all cell types are rings that unwind DNA by steric exclusion in which 3 the helicase ring only encircles the tracking strand, excluding the other strand outside the 4 ring. Steric exclusion mediated unwinding enables helicase rings to bypass blocks on the 5 strand that is excluded from the central channel. Unlike other replicative helicases, 6 eukaryotic CMG encircles duplex DNA at a forked junction and is stopped by a block on the 7 non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential 8 replication protein unique to eukaryotes, binds CMG and enables the replisome to bypass 9 blocks on the non-tracking strand, implying that Mcm10 isomerizes the CMG-DNA complex 10 to position only one strand through the central channel. A similar CMG-DNA isomerization is 11 needed at the origin for head-to-head CMGs to bypass one another during formation of 12 bidirectional replication forks. 13 14 Introduction 15 16 The replication of cellular DNA requires use of a helicase to separate the strands. The 17 replicative helicase in all domains of life is a circular hexamer. There are four superfamilies 18 of hexameric helicases, SF3-6, that assort into two main groups, the bacterial helicases 19 (SF4,5) that have ATP sites derived from the RecA fold and eukaryotic/archaeal helicases 20 (SF3,6) that have ATP sites derived from the AAA+ fold (Singleton et al., 2007). In all cases, 21 the subunits of hexameric helicases are composed of two major domains, giving them the 22 appearance of two stacked rings, an N-tier ring and C-tier ring; the motors are contained in 23 the C-tier ring. The RecA based bacterial helicases track 5’-3’ on DNA with the C-tier motors 24 leading the N-tier, as determined by crystal structures of bacterial Rho (SF5) and DnaB 25 (SF4) while eukaryotic helicases track 3’-5’ with the N-tier ahead of the C-tier as 26 demonstrated by structures of bovine papilloma virus E1 (SF3) and S. cerevisiae CMG (SF6) 27 (Enemark and Joshua-Tor, 2006; Georgescu et al., 2017; Itsathitphaisarn et al., 2012; 28 Thomsen and Berger, 2009). 29 30 Hexameric helicases are thought to act by encircling only one strand of DNA upon which 31 they track and exclude the non-tracking strand to the outside of the ring, thereby acting as a 32 wedge to split DNA in a process often referred to as steric exclusion and illustrated in 33 Figure 1A (Bell and Labib, 2016; Enemark and Joshua-Tor, 2008; Lyubimov et al., 2011). 34 Whether a helicase functions by steric exclusion is determined by biochemical experiments 35 that place a bulky block on one or the other strand of the duplex. A block placed on the non36 tracking strand (i.e. the strand that is excluded from the central channel) does not inhibit 37 helicase unwinding, while a block placed on the tracking strand stops the helicase because 38 the bulky block cannot fit through the central channel. 39 40 The eukaryotic helicase is the Mcm2-7 heterohexamer which requires five additional 41 accessory proteins Cdc45 and the four subunit GINS complex – for full activity. This 1142 subunit assembly is referred to as CMG (Cdc45, Mcm2-7, GINS) (Ilves et al., 2010; Moyer et 43 al., 2006). The recent cryoEM 3D structure of S. cerevisiae CMG helicase at a DNA replication 44 fork shows a unique DNA binding feature (Figure 1B) (Georgescu et al., 2017). Instead of 45 encircling only ssDNA, the N-tier of CMG encircles dsDNA and the DNA unwinding point is 46 buried inside the central channel; the unwound leading strand then proceeds through the 47 central channel into the C-tier motor domain. The lagging strand is not visualized in the 48 structure, indicating mobility, and is proposed to bend back out of the center of the ring. The 49 dsDNA is held at a 28o angle to the central channel, surrounded by the zinc fingers at the 50 . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/146837 doi: bioRxiv preprint first posted online Jun. 6, 2017;