Origin Firing Regulations to Control Genome Replication Timing

Abstract

Complete genome duplication is essential for genetic homeostasis over successive cell generations. Higher eukaryotes possess a complex genome replication program that involves replicating the genome in units of individual chromatin domains with a reproducible order or timing. Two types of replication origin firing regulations ensure complete and well-timed domain-wise genome replication: (1) the timing of origin firing within a domain must be determined and (2) enough origins must fire with appropriate positioning in a short time window to avoid inter-origin gaps too large to be fully copied. Fundamental principles of eukaryotic origin firing are known. We here discuss advances in understanding the regulation of origin firing to control firing time. Work with yeasts suggests that eukaryotes utilise distinct molecular pathways to determine firing time of distinct sets of origins, depending on the specific requirements of the genomic regions to be replicated. Although the exact nature of the timing control processes varies between eukaryotes, conserved aspects exist: (1) the first step of origin firing, pre-initiation complex (pre-IC formation), is the regulated step, (2) many regulation pathways control the firing kinase Dbf4-dependent kinase, (3) Rif1 is a conserved mediator of late origin firing and (4) competition between origins for limiting firing factors contributes to firing timing. Characterization of the molecular timing control pathways will enable us to manipulate them to address the biological role of replication timing, for example, in cell differentiation and genome instability.

Keywords: eukaryotic origin firing; origin firing regulation; origin firing timing; replication timing.

Figure 1

Proper control of origin firing is required for complete genome duplication. (A) Well-placed origins and efficient firing are required for complete DNA replication (1.) Replication problems can arise if not enough origins fire (2.) or if origins are misplaced (3.) Such misregulations can result in gaps in the replicating genome that are too big to be replicated by two forks during an S-phase. (B,C) Schematic representation of the dominos-like model in which replication spreads from the first replicating domain to its neighbours. This replication spreading is blocked at timing transition regions (C). Timing of the later domain at timing transition regions (TTRs) requires a new timing signal.

Figure 2

Molecular mechanisms of origin firing. Schematic representation of origin firing—a pre-RC (A) being converted into two active bi-directional replisomes (H)—and the steps required (B–G), as detailed in the main text. The blue box indicates pre-IC formation (B–F). Some details shown are hypothetical. For example, we speculate that two pre-IC complexes are required to originate two bi-directional forks but other models are also possible.

Figure 3

Two models of origin firing control to determine replication timing. (A) In model 1, origin firing timing is determined by chromatin organization. Different architectural chromatin units, depending on their structure, allow access/activate or block/inactivate firing factors, defining the timing of origin firing. Origins within a unit fire nearly synchronously when firing is allowed. (B) In model 2, origin firing timing is regulated at the origin level. This postulates that origins in a cluster fire synchronously because they are regulatorily coupled. Activating and inhibiting regulations define whether a cluster of origins fires or not at a given time.

Figure 4

Origin firing pathways that control replication timing of yeast chromosomes. Work in fission and budding yeast has revealed several molecular pathways that determine whether a chromosomal region replicates early (green) or late (orange). Ctf19/Swi6 promotes early replication of the centromere, while Fkh1/2 are involved in the early replication of some regions of the chromosome arm. Rif1 and/or Taz1 and/or shelterin dependent regulations ensure the late replication timing of the telomeric, sub-telomeric and some chromosomal arm regions. Other uncharacterised mechanisms for early or late replication timing regulations exist and are indicated with a question mark.