The properties of Mcm10, its role in Cdc17 stability and association at the fork (33
), and its interaction with the MCM helicase all suggest that Mcm10 plays a pivotal role in physically linking and coordinating the activities of polymerase α primase and the MCM helicase. We reasoned that suppression of the loss of this linker function would involve either restoration of Mcm10 function or recruiting another pathway to coordinate the polymerase and helicase activities. The finding that mcm2
suppressors do not restore the interaction of Mcm2 with the mutant Mcm10 protein or stabilize the mutant Mcm10 protein suggests that the latter is more likely. In achieving this end, the mutant helicase has to play a critical role. Although no detectable physiological defect is observed in the mcm2
suppressor other than the mild mcm
defect, the archaeal MCM helicase bearing the suppressor mutations invariably showed a compromised helicase activity. This result suggests that the altered helicase activity is critical for the suppression of the conditional lethality of mcm10
What are the phenotypes associated with the mcm10
conditional lethality? They should be phenotypes that are also suppressed by the mcm2
suppressors. We showed that mcm2
suppresses the replication fork pausing phenotype as well as HU and MMS sensitivity of mcm10
. Furthermore, it suppresses the synthetic growth defects of the loss of Sgs1, Exo1, or Srs2, a cohort of DNA helicases/nucleases, in mcm10
strains. These helicases/nucleases are involved in DSB repair (DSBR) in two capacities: (i) through resolution of aberrant fork structures to prevent DSB formation, and (ii) through resection of DSBs after their formation (19
). In contrast, mcm2
fails to suppress the synthetic growth defects of mcm10 mre11
or mcm10 rad50
strains. Mre11 and Rad50 are the major proteins involved in all DSB repair. The differences in requirement of these DSBR proteins in the suppression of mcm10
suggest that mcm2
is able to either substitute for Sgs1, Exo1, and Srs2 in the repair of their DSB substrates or prevent the formation of these substrates. The roles of Sgs1 and Exo1 are well defined in the DSB repair pathway, so it is doubtful that a defective MCM helicase could carry out their functions, especially those involving nuclease activities. A more likely scenario is that mcm2
is preventing the formation of aberrant fork structures and thereby abrogates the need for these helicases/nucleases. Under this scenario, we imagine that reduced activity of the helicase prevents the formation of a subset of DNA damage due to aberrant fork structures caused by the instability of Mcm10-1.
Mec1 is important for preventing dissociation of fork components, polymerase α in particular, when replication forks stall under replication stress (9
). It was shown that Mec1, rather than Rad53, plays a key role in maintaining the association of Polα with the replication fork when forks stall. Therefore, the Mec1-dependent stabilization of Cdc17 in mcm10 mcm2
cells suggests that preventing fork collapse is a key factor in preserving the viability of cells despite loss of Mcm10 function. Though it is possible that the Mcm2 suppressor stabilizes Cdc17 by acquiring the ability to interact directly between Polα and the helicase, two-hybrid analysis of Cdc17 with either wild-type or mutant Mcm2 does not support this hypothesis (data not shown). We believe that normally Mcm10 may stabilize Polα by direct interaction, but in the event that Mcm10 fails to carry out this function, alternative pathways may be evoked to substitute for this critical activity. An alternative explanation is that the stability of Polα depends on fork integrity as a whole rather than interaction with any particular protein and that cells are multifaceted in maintaining the integrity of the replication fork under normal or stress conditions.
In summary, our study suggests that reduced MCM helicase activity rendered by the mcm2 suppressor mutation is able to mediate fork stabilization by activating the checkpoint pathway and coordinating the helicase and polymerase activities in the absence of Mcm10. A model of how mcm2 may suppress mcm10 is shown in Fig. . In a normal replication fork, Mcm10, by interaction with both Polα and the MCM helicase, coordinates the polymerizing and unwinding activities on the lagging strand (Fig. ). In the mcm10 mutant, Mcm10 is unstable at 37°C, resulting in the decoupling of Polα from the helicase. Polα released from chromatin is destabilized. The uncoordinated unwinding and polymerizing activities expose extensive ssDNA (Fig. ), especially on the lagging strand, resulting in fork collapse and other damage that cannot be rescued by checkpoint-activated repair (Fig. ). We imagine that mcm2 suppresses the mcm10 conditional lethality by preventing such irreversible damage. The suppressor mutations may alter the rate of helicase unwinding to the extent that ssDNA accumulation is reduced and fork collapse is diverted; however, coordination between the unwinding and polymerizing activities may still be imperfect. As a result, chronic activation of checkpoint response in mcm10 mcm2 (Fig. ) by persistent low-level ssDNA exposure works in the favor of the faulty replication fork by stabilizing it (Fig. ). In other words, we propose that the loss of physical stabilization at the fork caused by the unstable Mcm10 can be compensated for by a mechanistic stabilization that results from the compromised helicase and the activated checkpoint proteins to coordinate the lagging strand synthesis. This hypothesis points to the dynamics of fork components in adapting to the defects of one another and the integration of different cellular pathways, such as replication, repair, and checkpoints, to maintain the integrity of the genome.
FIG. 7. Model of mechanism by which mcm2 suppresses mcm10 conditional lethality. (A) Mcm10 couples helicase to Polα primase under normal replication conditions. (B) Degradation of Mcm10 causes dissociation of Polα from chromatin and failure to (more ...)