A bit more about licensing of replication forks in eukaryotic cells

BIOC/MCB 568 -- Fall 2010
John W. Little--University of Arizona

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Since eukaryotic cells have very many origins of replication, it is important to ensure that all of them fire once and only once per cell cycle. This is controlled by a mechanism termed "licensing". The critical event in licensing appears to be loading of the MCM complex (the eukaryotic homologue of DnaB, the replicative helicase) at the origin of replication. Loaded MCM forms a ring through which the DNA passes, so that MCM remains stably bound. Two MCM rings are apparently bound at an origin, and will travel in opposite directions with the two replication forks that move in either direction away from the origin during S phase. Importantly, the mechanism that loads MCM can only take place during late M phase and in G1 phase of the cell cycle, but not at other stages.

DNA replication occurs during S phase. Three events combine to carry out the second phase of the licensing reaction. First, as stated, MCM can't be loaded onto origins; only pre-loaded MCM molecules are active. Second, other events occur which lead to initiation of replication, followed by the forks moving away from the origin. Third, the pre-loaded MCM molecules move away from the origin, at the front of the forks, so that they cannot return to the origin. Hence, although replication creates two daughter origins at the location of the parental origin, MCM can't be loaded at the daughter origins, so initiation occurs once and only once.

The mechanisms that control these events differ in yeast and in metazoa (multi-cellular organisms). The details aren't important for our purposes, but here's a little bit of detail if you're interested. In yeast, the mechanism relies mostly on changes in protein phosphorylation that occur at different stages in the cell cycle (this phosphorylation is carried out by protein kinases termed CDK's, for cyclin-dependent kinases; as the name implies, their activity is controlled by the presence of other proteins termed "cyclins", which are present at low levels early in G1 and at high levels at the start of S phase--see a Cell Biology text for details). Changes in phosphorylation allow or prevent MCM loading and initiation of replication. In metazoa, the major point of control is a protein called Cdt1. A more detailed discussion of licensing, including these mechanisms, is in a review by Blow and Dutta.  Note that their use of the term "clamp loader" differs from the usual use of loading the sliding clamp at a replication fork.