Hsk1 ((TOP))

We previously reported that Cds1 kinase activation by HU depends on Hsk1 function.20 Therefore, we have examined the effect of hsk1-89 mutation on the hyperphosphorylation of Mrc1. At the permissive temperature (25C), the extent of HU-induced hyperphosphorylation was reduced (Fig. 1A, lane 4), and it was almost completely eliminated at the non-permissive temperature (30C) in hsk1-89 (Fig. 1B, lane 5). The mobility-shift was only partially lost in rad3 cells (Fig. 1B, lane 9),23 as compared to hsk1-89 cells. It is known that the residual phosphorylation depends on Tel1.23

Wild-type Hsk1 but not kinase-negative or kinase-attenuated Hsk1 restores HU resistance or hyperphosphorylation of Mrc1 in hsk1-89. pREP 81 (Vec), pREP 81-Hsk1 wild-type (Hsk1wt), pREP 81-Hsk1 K129D (Hsk1-kd; kinase-dead) or pREP 81-Hsk1 KK129,130RS[KK-RS] (Hsk1-ka; kinase-attenuated) was transformed into hsk1+ (YM71) or hsk1-89 (KO147) cells. (A) Five-fold serial dilutions of exponentially growing cells harboring each plasmid indicated were plated on EMM supplemented with Uracil (EMMura) agar medium with or without thiamine and incubated at 25 or 30C for seven days as indicated. One set of plates (right part for each temperature) contained 8 mM HU. (B and C) The same set of cells were grown at 25C in EMMura with or without thiamine as indicated. For HU-treatment at 25C, 15 mM HU was added to the half of the culture and the other half was non-treated. The incubation was continued for 4 hrs at 25C. For HU-treatment at 30C, 25 mM HU was added to the half of the culture and the other half was non-treated. The incubation was continued for 3 hrs at 30C. The whole cell extracts were prepared and analyzed by western blotting to detect Mrc1 and Hsk1 proteins.

Hsk1

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Activation of Cds1 effector kinase in response to HU addition is severely compromised in hsk1-89 mutant at the non-permissive temperature.20 Activation of Cds1 kinase was assessed in other temperature-sensitive mutants (such as cdc19 and cdc20) defective in the processes of DNA replication. In these mutants, FACS profile of DNA contents suggested arrest in late S to G2 phase (data not shown). However, Cds1 kinase appeared to be still activated by HU in theses mutants at a nonpermissive temperature (Fig. 6B), suggesting that Hsk1 is specifically required for Cds1 kinase activation.

Cdc7 kinase, conserved through evolution, is known to be essential for mitotic DNA replication. The role of Cdc7 in meiotic recombination was suggested in Saccharomyces cerevisiae, but its precise role has not been addressed. Here, we report that Hsk1, the Cdc7-related kinase in Schizosaccharomyces pombe, plays a crucial role during meiosis. In a hsk1 temperature-sensitive strain (hsk1-89), meiosis is arrested with one nucleus state before meiosis I in most of the cells and meiotic recombination frequency is reduced by one order of magnitude, whereas premeiotic DNA replication is delayed but is apparently completed. Strikingly, formation of meiotic dsDNA breaks (DSBs) are largely impaired in the mutant, and Hsk1 kinase activity is essential for these processes. Deletion of all three checkpoint kinases, namely Cds1, Chk1, and Mek1, does not restore DSB formation, meiosis, or Cdc2 activation, which is suppressed in hsk1-89, suggesting that these aberrations are not caused by known checkpoint pathways but that Hsk1 may regulate DSB formation and meiosis. Whereas transcriptional induction of some rec genes and horsetail movement are normal, chromatin remodeling at ade6-M26, a recombination hotspot, which is prerequisite for subsequent DSB formation at this locus, is not observed in hsk1-89. These results indicate unique and essential roles of Hsk1 kinase in the initiation of meiotic recombination and meiosis. e24fc04721