Dormant origins, the licensing checkpoint, and the response to replicative stresses

Abstract

Only ∼10% of replication origins that are licensed by loading minichromosome maintenance 2-7 (MCM2-7) complexes are normally used, with the majority remaining dormant. If replication fork progression is inhibited, nearby dormant origins initiate to ensure that all of the chromosomal DNA is replicated. At the same time, DNA damage-response kinases are activated, which preferentially suppress the assembly of new replication factories. This diverts initiation events away from completely new areas of the genome toward regions experiencing replicative stress. Mice hypomorphic for MCM2-7, which activate fewer dormant origins in response to replication inhibition, are cancer-prone and are genetically unstable. The licensing checkpoint delays entry into S phase if an insufficient number of origins have been licensed. In contrast, humans with Meier-Gorlin syndrome have mutations in pre-RC proteins and show defects in cell proliferation that may be a consequence of chronic activation of the licensing checkpoint.

Figure 1.

The effect of fork stalling on completion of replication. A small segment of chromosomal DNA is shown with two efficient licensed origins red hexamers) plus an optional dormant origin (green hexamers). Fork stalling is indicated by a vertical purple bar. (A) One fork stalls, but all the intervening DNA is replicated by the fork originating at an adjacent origin. (B) A dormant origin is inactivated by a fork coming from the right. (C) Each of the two converging forks stall without a dormant origin between them. Replication cannot be completed because no new MCM2-7 complexes can be loaded onto DNA once S phase has begun. (D) Two converging forks stall, but a dormant origin between them allows replication to be completed.

Figure 2.

Model for how cells respond to low levels of replicative stress. Two adjacent clusters of origins (factories bounded by yellow circles) are shown on a single piece of DNA (black lines). Under normal circumstances (left), the upper factory is activated slightly earlier than the factory below, and each initiates three origins. Under low levels of replicative stress (right), replication forks are inhibited in the earlier replicating cluster, which promotes the firing of dormant origins as a direct consequence of stochastic origin firing. Replicative stress activates DNA damage checkpoint kinases, which preferentially inhibit the activation of the unfired later clusters/new factories.

Figure 3.

How oncogene activation might normally disrupt licensing control. Progression through the cell cycle (as shown in the lower bar) depends on sequential activation of the licensing system (green) and initiation activities (fuchsia). (A) In normal cells, there is no significant overlap between licensing and initiation activities. The transition from the licensing state to the initiation state occurs around the restriction point. (B) If an oncogene is constitutively expressed that acts upstream of the restriction point, cells may be unable to fully repress licensing activity during S phase, potentially leading to re-replication of DNA. (C) If an oncogene is constitutively expressed that acts downstream of the restriction point, cells may not be able to license a sufficiently large number of origins before progressing into S phase, potentially leading to incomplete replication of DNA. See Blow and Gillespie (2008) for more discussion of these potential effects.

Figure 4.

The licensing checkpoint. A piece of DNA containing several origins is denoted by the black line. (A) When licensing occurs, ORC, Cdc6, and Cdt1 promote the loading of MCM2-7 (red hexamers) onto DNA. This allows normal activation of Cdk4/6 and Cdk2 kinases, and subsequent activation of E2F by Rb phosphorylation. (B) When the licensing is inhibited, the licensing checkpoint is activated, causing suppression of cyclin D transcription and activation of p53, resulting in induction and Cip1 and Kip1 and inhibition of activating Cdk2 phosphorylation.