In general, two replication forks emerge from an activated origin of replication, traveling in opposite directions.
The Hat1p association with origin DNA is analogous to that observed previously with a well established initiation cofactor, Cdc45p, which also advances with replication forks after recruitment to and release from an activated origin [ 5, 6].
S phase was significantly extended in all mutants, but the genome-wide replication program was hardly altered: the identity of activated origins and their relative activation times (or efficiencies) remained essentially the same as those in wild-type.
Since PREP1 down-regulation induces a decrease in the inter-origin distance (Fig. 2C,D), and considering constant processivity of DNA polymerases in siPREP1 and siLuc cells, altogether our data strongly suggest an increase in simultaneously activated origins within LRRs and/or TTRs, which normally show reduced replication origin activity23.
These include a local replication enhancer that activates origin firing at DAFC-66D (Orr-Weaver et al. 1989), ORC-independent origin firing at DAFC-34B (Kim and Orr-Weaver 2011), and local repression of ORC binding and origin activity at DAFC-22B (Kim et al. 2011).
These findings support a model in which replication timing is the result of competing signals, which may determine the availability of replication factors to activate origins.
A large body of evidence has revealed that this complex pathway, once activated, regulates origin firing, cell cycle arrest, stabilization, and resumption of stalled forks [ 39– 41].
The possible involvement of the replication checkpoint in the scaling phenotype was suggested [ 18, 19, 22], since deletion of either mec1 or rad53 activated late origins that were inhibited by HU treatment [ 23– 26].
Furthermore, expression of WT Orc1-1 driven by its native promoter from episomes, while resulting in origin occupancy (as adjudged by ChIP), fails to activate the origin, presumably due to hydrolysis of ATP in the cell prior to origin binding.
Altogether, this study provides new insights into the mechanisms that activate the origin of replication in eukaryotes by identifying a role for the Mcm2-7 helinase in the processes of strand selection and, probably, origin melting.
Three events must occur in order to activate the origin of replication in eukaryotes: the two Mcm2-7 rings must separate; each ring must trap the leading strand template; and each ring must eject the lagging strand template.
