How dormant origins promote complete genome replication

Abstract

Many replication origins that are licensed by loading MCM2-7 complexes in G1 are not normally used. Activation of these dormant origins during S phase provides a first line of defence for the genome if replication is inhibited. When replication forks fail, dormant origins are activated within regions of the genome currently engaged in replication. At the same time, DNA damage-response kinases activated by the stalled forks preferentially suppress the assembly of new replication factories, thereby ensuring that chromosomal regions experiencing replicative stress complete synthesis before new regions of the genome are replicated. Mice expressing reduced levels of MCM2-7 have fewer dormant origins, are cancer-prone and are genetically unstable, demonstrating the importance of dormant origins for preserving genome integrity. We review the function of dormant origins, the molecular mechanism of their regulation and their physiological implications.

Figure 1. Ensuring precise chromosome replication

A small segment of chromosomal DNA is shown, consisting of 3 domains each replicated from three replication origins. The domain is shown at different stages of the cell cycle: G1, early-, mid- and late-S phase and G2; a whole chromosome containing the chromosomal segment is shown in mitosis. A), The DNA is under-replicated as a consequence of origins in the middle cluster failing to fire. As sister chromatids are separated during anaphase, the chromosome is likely to be broken near the unreplicated section. B) Origins are correctly used and chromosomal DNA is successfully duplicated. C) One of the origins fires for a second time in S phase. The local duplication of DNA in the vicinity of the over-firing origin represents an irreversible genetic change and might be resolved to form a tandem duplication.

Figure 2. The licensing cycle

A small segment of chromosomal DNA that encompasses three replication origins is shown. At the end of mitosis (M), the replication licensing system is activated (light green), which causes MCM2-7 complexes (blue hexamers) to be loaded onto potential replication origins (origin licensing). The licensing system is turned off at the end of G1. During S phase, some MCM2-7 complexes are activated as helicases as origins fire (pink hexamers). MCM2-7 are removed from replicated DNA, either during passive replication of unfired origins, or at fork termination. In this way, replicated DNA cannot undergo further initiation events until passage through mitosis.

Figure 3. The effect of fork stalling on completion of replication

A small segment of chromosomal DNA is shown with either 2 or 3 licensed origins. MCM2-7 complexes at unfired origins are shaded blue, MCM2-7 complexes activated as replicative helicases are shaded pink. Irreversibly stalled replication forks are marked with a red ‘X’. A) One fork stalls, but all the intervening DNA is replicated by the fork originating at an adjacent origin. B) Each of the two converging forks stall. Replication cannot be completed because no new MCM2-7 complexes can be loaded onto DNA once S phase has begun. C) A dormant origin is inactivated by a fork coming from the left. D) Two converging forks stall, but a dormant origin between them allows replication to be completed.

Figure 4. Stochastic origin firing within a single cluster

Example of the computer model showing how stochastic origin firing leads to dormant origin activation if fork speed is slowed. A) A cartoon of the modelling process, with initial origin licensing, followed by repeated steps of initiation and elongation. During each step, a licensed origin undergoes a random test, to determine whether it fires. Once an origin has fired, replication forks proceed away from it, as shown by the arrows. If a fork passes over an unfired origin (passive replication of a licensed origin), the origin is inactivated. In the cartoon, two of the 5 origins have fired and one has been passively replicated. Arrows show direction of fork movement. B) Example output of the computer model where 16 licensed origins were randomly spaced on a 250 kb origin cluster (x axis). Each origin was assigned an initiation probability randomly distributed around a mean of 0.00508 per step. S phase was then enacted in steps of 25 seconds (y axis). Initiation events are marked by dark circles, passive replication is marked by faint circles and fork progression is represented by the lines. Line peaks represent termination events. Two simulations using identical origin parameters are shown: in red where forks proceed at a normal speed (20 nt/sec) and in blue where forks have been slowed to 5 nt/sec. The pattern of origin usage is also shown on the linear DNA molecules at the top. Sample data taken from reference [41].

Figure 5. Model for how cells respond to low levels of replicative stress

A) Two adjacent clusters of origins (factories bounded by green 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. B) A single piece of DNA (black line) is shown with two converging forks that have stalled (red bars). If a dormant origin is activated between them, replication can be rapidly rescued (left). If there is no dormant origin firing between the stalled forks (right), the DNA damage response can lead to recombination or induction of apoptosis. Reproduced, with permission, from reference [55].