Replicating Large Genomes: Divide and Conquer

Abstract

Complete duplication of large metazoan chromosomes requires thousands of potential initiation sites, only a small fraction of which are selected in each cell cycle. Assembly of the replication machinery is highly conserved and tightly regulated during the cell cycle, but the sites of initiation are highly flexible, and their temporal order of firing is regulated at the level of large-scale multi-replicon domains. Importantly, the number of replication forks must be quickly adjusted in response to replication stress to prevent genome instability. Here we argue that large genomes are divided into domains for exactly this reason. Once established, domain structure abrogates the need for precise initiation sites and creates a scaffold for the evolution of other chromosome functions.

Figure 1

The distinct scales of DNA replication regulation in eukaryotes. A) Replication origin regulation. The origin recognition complex (ORC) binds at all potential replication origins that become licensed by the CDT1-dependent recruitment of MCM-helicase complex (a double hexamer of the subunits MCM2-7) to form the pre-RC. All potential origins are licensed but only ~10% are activated. Additional factors are recruited, followed by DDK phosphorylation of distinct residues of the MCM complex that trigger the helicase activity, splitting the MCM hexamers and starting the bidirectional unwinding of the DNA strands. For simplicity, the depiction does not include all components of the pre-RC and pre-IC. For a detailed description see (Yeeles et al., 2015). B) DNA replication regulation at the replication domain scale. Synchronized firing of clusters of origins activated either early or late during S-phase partition the chromosomes into replication domains. C) DNA replication at the nuclear scale and its regulation during the cell cycle. RDs are segregated within the nucleus in such way that early replicating segments of the chromosomes occupy the nuclear interior while the late RDs are preferentially located close to the nuclear and nucleolar (N) periphery. Origin licensing occurs only from M to G1, then CDT1 is rapidly degraded and sequestrated by geminin blocking origin re-licensing although additional inhibitors might repress pre-RC activation during the G1-S transition (Sasaki et al., 2011). Then an induction of DDK/S-CDK in S-phase activate the pre-IC to initiate replication. The timing decision point (TDP) occurs early during G1 and precedes origins selection (ODP).

Figure 2

Heterogeneity in replication origin selection and regulation of replication activation under stress. A) Multiple potential replication origins are licensed within each RD. Distinct cells within the same population use different origins under normal conditions. B) Regulation of origin activation under replication stress. When replication forks are stalled, checkpoint responses are induced to promote the activation of dormant origins within the RD while inhibiting the firing of origins in RDs that are activated later in S-phase (Gilbert, 2007).

Figure 3

G-quadruplexes (G4) and replication origin specification. A) G4 structure. Rings of guanine quartets are stacked on top each other and stabilized by hydrogen bonds. B) G4 might cooperate with additional cis elements (such as NFRs or AT-rich regions) to specify the more efficient location of replication initiation (modified from (Valton et al., 2014). C) Gene expression can influence origin selection by generating R-loops that enhance G4 formation or “pushing” the pre-RC towards G4 motifs.

Figure 4

Dynamic changes in replication regulation during development. A) RT changes during development defining distinct classes of RDs: constitutive domains (replicating always either early or late) and developmental regulated domains that switch between early and late replication. B) Developmentally regulated domains are distinct from constitutive RDs.