Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jul 21;357(6348):314-318.
doi: 10.1126/science.aan0063.

Bidirectional Eukaryotic DNA Replication Is Established by Quasi-Symmetrical Helicase Loading

Affiliations
Free PMC article

Bidirectional Eukaryotic DNA Replication Is Established by Quasi-Symmetrical Helicase Loading

Gideon Coster et al. Science. .
Free PMC article

Abstract

Bidirectional replication from eukaryotic DNA replication origins requires the loading of two ring-shaped minichromosome maintenance (MCM) helicases around DNA in opposite orientations. MCM loading is orchestrated by binding of the origin recognition complex (ORC) to DNA, but how ORC coordinates symmetrical MCM loading is unclear. We used natural budding yeast DNA replication origins and synthetic DNA sequences to show that efficient MCM loading requires binding of two ORC molecules to two ORC binding sites. The relative orientation of these sites, but not the distance between them, was found to be critical for MCM loading in vitro and origin function in vivo. We propose that quasi-symmetrical loading of individual MCM hexamers by ORC and directed MCM translocation into double hexamers acts as a unifying mechanism for the establishment of bidirectional replication in archaea and eukaryotes.

Figures

Figure 1
Figure 1. A single ORC binding site is insufficient for optimal MCM loading and origin function
(A) MCM recruitment and loading are unaffected by a luciferase tag and are defective in Mcm3-13. (B) Untagged WT MCM does not rescue the loading defect of Mcm3-13. (C) A synthetic ORC binding site does not support plasmid replication in vivo, even in the presence of Abf1 binding sites (D) or poly(dA) stretches of increasing length (E), assayed by the formation of yeast colonies after transformation. (F,G) ORC binding to the synthetic site is ATP dependent and comparable to natural origins. (H) The presence of chloride during loading (80 mM KCl) drives sequence specific MCM loading and reveals that natural origins are more efficient than a single synthetic site.
Figure 2
Figure 2. Two ORC sites are necessary and sufficient for maximal MCM loading and origin activity
(A) ORC binding, (B) MCM recruitment and (C) MCM loading onto synthetic substrates containing one or two ORC binding sites in all possible orientations. in vivo origin activity of synthetic two-site substrates in the absence (D) or presence (E) of Abf1 binding sites. (F) Three base pair changes that create a second ORC site are sufficient to convert the Poly(dA) substrates from Figure 1E into active origins.
Figure 3
Figure 3. Natural origins employ one high affinity site and additional secondary sites
(A) Salt sensitivity of one-site loading suggests the usage of a second non-specific site. The indicated amount of salt was present during loading. All reactions were subsequently washed with high salt (1M NaCl). (B) One-site loading is more sensitive to competitor DNA than two-site loading. (C) MCM recruitment and loading as a function of ORC concentration. Data is plotted as mean ±SEM. (D) Natural origins harbor secondary ORC binding sites. (E) MCM loading with synthetic versus natural origins, as well as deletion mutants (See Fig. S6). (F) Mutations in secondary sites impair MCM loading in ARS600.1 and ARS1216.
Figure 4
Figure 4. Two-site loading exhibits flexible spacing and is sensitive to an intervening roadblock
(A) Synergistic loading and (B) origin activity exhibit flexible inter-site spacing. (C) A covalent DNA-protein roadblock between two-sites inhibits synergistic loading. (D) Only the complete roadblock reaction leads to a block in loading. (E) Proposed model for MCM loading. See text for details.

Similar articles

See all similar articles

Cited by 24 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback