Checkpoint-Independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 Kinase

ABSTRACT

Mrc1 is a conserved checkpoint mediator protein that transduces the replication stress signal to the downstream effector kinase. The loss of mrc1 checkpoint activity results in the aberrant activation of late/dormant origins in the presence of hydroxyurea. Mrc1 was also suggested to regulate orders of early origin firing in a checkpoint-independent manner, but its mechanism was unknown. Here we identify HBS (Hsk1 bypass segment) on Mrc1. An ΔHBS mutant does not activate late/dormant origin firing in the presence of hydroxyurea but causes the precocious and enhanced activation of weak early-firing origins during normal S-phase progression and bypasses the requirement for Hsk1 for growth. This may be caused by the disruption of intramolecular binding between HBS and NTHBS (N-terminal target of HBS). Hsk1 binds to Mrc1 through HBS and phosphorylates a segment adjacent to NTHBS, disrupting the intramolecular interaction. We propose that Mrc1 exerts a “brake” on initiation (through intramolecular interactions) and that this brake can be released (upon the loss of intramolecular interactions) by either the Hsk1-mediated phosphorylation of Mrc1 or the deletion of HBS (or a phosphomimic mutation of putative Hsk1 target serine/threonine), which can bypass the function of Hsk1 for growth. The brake mechanism may explain the checkpoint-independent regulation of early origin firing in fission yeast.

KEYWORDS:

• DNA replication timing
• Mrc1
• Hsk1
• intramolecular interaction
• replication checkpoint

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00355-16.

ACKNOWLEDGMENTS

This work was supported by JSPS KAKENHI (grant-in-aid for scientific research [A] [grant numbers 23247031 and 26251004] and grant-in-aid for scientific research on priority areas [“noncoding RNA” and “genome adaptation,” grant numbers 24114520 and 25125724, respectively] to H.M. and grant-in-aid for scientific research [C] [grant number 24570205] to S.M.) and by the Naito Foundation Continuation Subsidy for Outstanding Projects (to H.M.).

S.M., Y.K., and H.M. conceived of and designed experiments. S.M. constructed strains and performed and analyzed all S. pombe experiments. Y.K. performed ChIP-quantitative PCR and ChIP-seq. M.S. and H.M. performed biochemical experiments. M.H. performed two-dimensional gel electrophoresis and ChIP-quantitative PCR. K.U. aided in the initial phase of this project with the construction of strains and biochemical analysis. R.F., N.K., and H.M. constructed some plasmids and purified Mrc1 proteins. S.M. and H.M. wrote the paper.

We declare that we have no conflict of interest.