DNA replication origins retain mobile licensing proteins

Abstract

DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms.

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence

“…1B). Consistent with previous results (21,22), ORC-Cdc6 displayed diffusive behavior at non-ARS1 sites until it dissociated or encountered the ARS1 site, where the rapid diffusion behavior halted and, instead, ORC-Cdc6 resided stably (Fig. 1B).…”

“…1B), as well as Cdc6 and Cdt1. To generate fluorescently labeled ORC for single-molecule visualization, we site-specifically attached a Cy3 fluorophore to the N terminus of the Orc1 subunit via a 12-residue peptide tag (S6), much smaller than the Halo-tag used in a recent single-molecule study (22). A single λARS1 molecule was tethered between a pair of streptavidin-coated beads in the microfluidic chamber of a dual-trap optical tweezers instrument also equipped with multicolor confocal fluorescence microscopy (24, 25) (Supplementary Fig.…”

“…Single-molecule studies using reconstituted replication proteins have greatly aided our understanding of origin recognition and pre-RC formation (20)(21)(22). However, these studies have not examined the effect of nucleosomes on ORC activity.…”

A Helicase-tethered ORC Flip Enables Bidirectional Helicase Loading

“…As described above, for a number of origins for which B2 elements have been identified (including ARS1) this sliding would be required to expose the B2 element (in Figure 7, sliding to the left). A number of helicase-loading intermediates slide in vitro (Sánchez et al, 2021;Scherr et al, 2021), and experiments placing barriers to sliding at ARS1 support a role for sliding to identify second inverted ORC-binding sites (Coster and Diffley, 2017;Warner et al, 2017). Consistent with the argument that second recruitment only occurs when the sliding pre-M1O finds a second inverted ORC-binding site, helicase loading does not occur at artificial origins with a single strong ORC-binding site (Coster and Diffley, 2017).…”

A helicase-tethered ORC flip enables bidirectional helicase loading

“…As described above, for a number of origins for which B2 elements have been identified (including ARS1 ) this sliding would be required to expose the B2 element (in Figure 7 , sliding to the left). A number of helicase-loading intermediates slide in vitro ( Sánchez et al, 2021 ; Scherr et al, 2021 ), and experiments placing barriers to sliding at ARS1 support a role for sliding to identify second inverted ORC-binding sites ( Coster and Diffley, 2017 ; Warner et al, 2017 ). Consistent with the argument that second Mcm2-7 recruitment only occurs when the sliding pre-M 1 O finds a second inverted ORC-binding site, helicase loading does not occur at artificial origins with a single strong ORC-binding site ( Coster and Diffley, 2017 ).…”