Bidirectional replication from eukaryotic DNA replication origins requires the loading of two ringshaped 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 origins and synthetic 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 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 Eukarya.The motor of the eukaryotic replicative helicase -the heterohexameric MCM complex -is loaded onto replication origins as an inactive, head-to-head double hexamer (DH) during the G1 phase of the cell cycle (1-3). In S phase, the DH is converted into two active CMG (Cdc45-MCM-GINS) helicases (4-6), that nucleate assembly of the two bidirectional replisomes. MCM loading begins with the binding of ORC to DNA. ORC, together with Cdc6 and Cdt1, then recruits the first MCM hexamer (7,8). ATP binding by ORC, Cdc6 and MCM is required for this recruitment, and ATP hydrolysis by MCM then drives DH assembly (9, 10). But how the second hexamer is recruited and loaded in the correct position and orientation is unclear (Fig. S1A).The recruitment of the first MCM hexamer to ORC/Cdc6 is mediated by the C-terminus of Mcm3 (11). Based on single molecule approaches, it has been proposed that the second MCM hexamer is directly recruited by interaction with the first hexamer, aided by a second Cdc6 but not a second ORC molecule (8,12). If this were the case, point mutants in the Cterminus of that cannot be recruited as the first hexamer, should still be able to load as a second hexamer in the presence of wild type MCM. However, such mutants fail to load even when mixed with wild type MCM, suggesting that both hexamers must be able to bind ORC/Cdc6 for loading (11). It remains possible that Mcm3-13 loading still occurs, but at a level below detection limits. We therefore developed a more sensitive and quantitative assay for MCM loading based on a fusion of Mcm3 to luciferase (Fig. 1A and Fig. S2). The Mcm3-13 mutation had no effect on MCM complex stability (Fig. S1B (Fig. S1C). However, despite the improved sensitivity, no loading of Mcm3-13 was observed in the absence or presence of wild type MCM over a range of ratios ( Fig. 1B and Fig. S1D, E).Although this result suggested that both MCM hexamers must be able to bind ORC/Cdc6, budding yeast origins generally contain a single high affinity ORC binding site located at one end of a Nucleosome Free Region (NFR) (13). To test whether these features are sufficient for origin activity in vivo, we constructed synthetic sequences b...
SummaryLoading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs.
Accurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstitute replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We find that structure-prone repeats are sufficient to impair replication. Whilst template unwinding is unaffected, leading strand synthesis is inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats is rescued by replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats requires an extrinsic accessory helicase. DNA-induced fork stalling is mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress may also be applied to DNA-induced replication stalling.
MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (␥-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of ␥-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.
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