DDK links replication and recombination in meiosis

Meiosis begins with DNA replication followed by homologous recombination and nuclear divisions. Since programmed DNA double-strand breaks (DSBs) were discovered to initiate meiotic recombination, extensive studies have identified the players required for DSB formation and the chromosomal features that determine DSB frequency and distribution. However, the temporal control of DSB formation, especially coordination with preceding replication, is poorly understood.

A study previously revealed that the timing of DSB formation is regulated on a local rather than a cell-wide basis.¹ In that study, a late-replicating chromosomal region was created by deleting replication origins from the left arm of Saccharomyces cerevisiae chromosome III (chrIII). The strain exhibits a corresponding delay in DSB formation exclusively in the late-replicating arm, suggesting that the timing of DSB formation is dictated locally by replication. However, the mechanisms remained elusive.

In S. cerevisiae, meiotic DSB formation requires at least 10 proteins (DSB proteins). One of them, Mer2, is phosphorylated by Dbf4-dependent kinase (DDK: consisting of catalytic subunit Cdc7 and regulatory subunit Dbf4) and the phosphorylation is essential for DSB formation.^2,3 Mer2 phosphorylation is required for chromatin association of certain DSB proteins, but not for Mer2 binding to chromatin.^2,4 DSBs appear ∼90 minutes after the phosphorylation.³ These findings suggest that Mer2 phosphorylation is an initial step toward DSB formation. DDK is required for initiation of both mitotic and meiotic replication. The cellular DDK level is low early in meiosis and increases as meiosis progresses and the level of DDK required for meiotic replication is lower than that for DSB formation.³ This sensitivity difference to DDK can explain why replication precedes DSB formation, but not the local relationship between replication and DSB formation.

We hypothesized that Mer2 phosphorylation occurs preferentially in the wake of the replication fork so that an initial step toward DSB formation is triggered exclusively in chromosomal regions where replication is completed, thereby establishing temporal and spatial replication-DSB coordination (Fig. 1).⁵ To achieve this preferential phosphorylation, the cellular DDK level should be limiting and specifically targeted toward replicated chromatin.

Figure 1.

Top: S. cerevisiae Chromosome III with replication origins are shown. Origin deletion delays replication exclusively in the left arm and causes a corresponding delay in DSB formation (replication-DSB coordination). Bottom: DDK is recruited to the replication fork via Tof1/Csm3 and phosphorylates Mer2 to trigger DSB formation preferentially in the replicated chromosomal regions. Furthermore, DDK recruitment sequesters DDK from unreplicated regions. Figure adapted from the online graphical abstract for reference 5.

To test our model, we used the origin-deleted strain mentioned above for an assessment of replication-DSB coordination and eliminated the first prerequisite by overproducing DDK in meiosis. We found that upon DDK overproduction DSBs form with the same timing regardless of replication time difference, suggesting that the Mer2 phosphorylation occurs irrespective of replication timing and that, in wild-type meiosis, DDK must be limiting to establish replication-DSB coordination.

Next, we sought to examine the second prerequisite: the mechanism that recruits DDK to the replication fork. The Dbf4 homolog in Schizosaccharomyces pombe interacts with components of the fork protection complex (FPC).⁶ In S. cerevisiae FPC consists of Tof1, Csm3 and Mrc1 (Timeless, Tipin and Claspin in higher eukaryotes), and travels with moving replication forks,⁷ raising the possibility that FPC recruits DDK to the replication fork. We confirmed by immunoprecipitation that Tof1 interacts with Dbf4 in meiosis. Then, we deleted TOF1 to see if it affects replication-DSB coordination. Indeed, tof1Δ showed uncoupling of DSB formation and replication, suggesting that tof1Δ disrupts replication-associated Mer2 phosphorylation which now occurs regardless of replication time when DDK reaches a higher level later in meiosis. Consistent with this prediction, tof1Δ exhibited a global delay in DSB formation in meiosis compared to wild-type and DDK overproducing strains. csm3Δ similarly showed disruption in replication-DSB coordination but mrc1Δ did not, indicating that the coordination requires Tof1 and Csm3 but not the replication checkpoint functions mediated by Mrc1. If Tof1 acts as a linker between the replication fork and DDK, artificially tethering DDK to the fork should bypass the Tof1 requirement. Indeed, when Dbf4 was fused to Cdc45, a component of the replication fork machinery, replication-DSB coordination was substantially rescued in tof1Δ. Taken together, we concluded that DDK recruitment to the replication fork through Tof1/Csm3 establishes replication-DSB coordination.

Finally, we asked if Mer2 phosphorylation is coordinated with local replication timing. Since chromatin association of Rec114 (another DSB protein) is dependent on Mer2 phosphorylation, we indirectly assessed Mer2 phosphorylation status by Rec114 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). As predicted, Rec114 ChIP showed delay exclusively in the origin-deleted left arm of chrIII. Genome-wide temporal profiles of Rec114 chromatin association and replication showed a correlation where early and late replicating chromosomal regions tend to show early and late Rec114 association respectively, and this correlation decreased in tof1Δ. Taken together, we conclude that the timing of Rec114 chromatin association (an indirect readout of Mer2 phosphorylation) is coordinated with local replication in a Tof1-dependent manner and that this coordination is not specific to artificial replication delay caused by origin deletion.

We identified a molecular mechanism which links replication and DSB formation (Fig. 1).⁵ The regulatory system we discovered is counterintuitive. When DDK executes a key event, its protein level is undetectably low. Remarkably, having such a low protein level is itself a prerequisite for the system to function. It is notable that the regulatory module we discovered is shaped by a ubiquitous kinase and widely conserved replisome components. Recognizing this surprising feature is a novel insight that is likely to be generally applicable for understanding how cellular processes can be restricted within replicated chromatin.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.