Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM

Abstract

In preparation for bidirectional replication, the origin recognition complex (ORC) loads two MCM helicases forming a head-to-head double hexamer (DH) around DNA 1,2. How DH formation occurs is debated. Single-molecule experiments suggest a sequential mechanism whereby ORCdependent loading of the first hexamer drives second hexamer recruitment 3. In contrast, biochemical data show that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites 4,5. We visualized MCM loading using time-resolved EM, to identify DH formation intermediates. We confirm that both hexamers are recruited via the same interaction between the MCM and ORC C-terminal domains, and identify the mechanism for coupled MCM loading. A first loaded hexamer locked around DNA is recognized by ORC, which unexpectedly engages the N-terminal homo-dimerization interface of MCM. In this configuration, ORC is poised to direct second hexamer recruitment in an inverted orientation, suitable for DH formation. Our data reconcile two apparently contrasting models. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Competition for MCM Loading at Origins Establishes Replication Timing Patterns

“…Notably, the MCM signal was largely independent of ACS directionality, as only 50% of higher-upstream-signal origins and 44% of higherdownstream-signal origins correlated with ACS directionality. This data supports the model where, once loaded, MCM is able to passively slide in either direction on double stranded DNA, consistent with previous in vitro findings (Remus et al, 2009;Miller et al, 2019).…”

“…The signal includes MCM double hexamer-sized reads (~68bp) as well as larger, nucleosome-sized reads (~146bp) (Figure 3a). In contrast to previous genome-wide reports (Belsky et al, 2015), but in agreement with recent in-vitro cryo-EM structures (Miller et al, 2019), we also observe MCM signal in the nucleosome-depleted region (NDR) of origins. This observation was dependent on the degree of MNase digestion, as Replicate #1, which had a lower degree of digestion, lacked the smaller fragments, in agreement with similar studies involving transcription factors ( Figure S4) (Henikoff et al, 2011).…”

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

“…More recent attempts in the framework of cryo-EM and single-particle analysis have traced structural changes over time in processes that proceed over several seconds and even minutes. This was only possible because the studied processes were slower than the time required to prepare a cryo-EM sample grid by the standard method of manual application, followed by automated blotting and plunge-freezing [14][15][16][17] . Dynamic structural states could also be resolved by freeze-trapping samples, pre-incubated at different temperatures 18 .…”

Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

“…Although bidirectional origin firing has long been recognized as a universal feature of chromosomal DNA replication in all domains of life (Huberman and Riggs, 1968;Prescott and Kuempel, 1972), how pairs of oppositely oriented replication forks are established at chromosomal origins remains poorly understood. In eukaryotes, two copies of the replicative DNA helicase, Mcm2-7, are loaded as a stable double-hexameric complex around double-stranded DNA (dsDNA) at the origin (Evrin et al, 2009;Miller et al, 2019;Remus et al, 2009). Mcm2-7 comprise six related proteins of the AAA+ family of ATPases that assemble into a hexameric ring with defined subunit order.…”

“…Intriguingly, the two hexamers in a Mcm2-7 double hexamer (DH) associate in a head-to-head configuration, thus providing a platform for the establishment of oppositely oriented sister replisomes. (Abid Ali et al, 2017;Evrin et al, 2009;Li et al, 2015;Miller et al, 2019;Noguchi et al, 2017;Remus et al, 2009). However, Mcm2-7 DHs are catalytically inactive and require the regulated association of the essential helicase co-factors Cdc45 and GINS to form two active replicative DNA helicase complexes, termed CMG (Cdc45-MCM-GINS), which encircle single-stranded DNA (ssDNA) during unwinding (Bell and Labib, 2016).…”