ge in chromatin and transduces a signal to an effecter kinase, Chk1, by phosphorylating it. The requirement for rad3 and chk1 for the survival of the asf1-33 mutant suggested that the Chk1 pathway was activated in these cells. We therefore examined whether Chk1 is phosphorylated in the asf1-33 mutant at 36uC by testing for a phosphorylation-induced mobility shift in Chk1 using phosphatebinding tag in a phosphate affinity SDSPAGE. In this assay, phosphorylated proteins are captured by Phos-tagTM in the SDS-PAGE gel during electrophoresis and their mobility is super-shifted. Using this method, phosphorylated-Cds1 protein was identified but there was no evidence for Chk1 phosphorylation. We then changed the acrylamide:bisacrylamide ratio from 37.5:1 to 200:1 in order to more clearly separate phosphorylated and non-phosphorylated Chk1. Using these buy VX765 conditions, we were able to detect the mobility shift of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22179956 phosphorylated Chk1 in the asf1-33 mutant at 36uC by western blotting. In contrast, Cds1, a DNA replication checkpoint factor, was not phosphorylated in the asf1-33 mutant at 36uC. Furthermore, we found that a phosphorylation-deficient mutant of chk1 showed a similar phenotype to the asf1-33 Dchk1 mutant. Taken together, these results indicated that a DNA damage checkpoint, but not a DNA replication checkpoint, was activated in the asf1-33 mutant at 36uC. We next examined the drug sensitivity of the asf1-33 mutant at different temperatures. At the semi-restrictive temperature, the asf1-33 mutant was sensitive to the DNA damaging agent methyl methanesulfonate . This result is consistent with the requirement of DNA damage checkpoint factors for survival and cell cycle checkpoint activation in the asf1-33 mutant. In contrast, the asf1-33 mutant was not sensitive to hydroxyurea at 34uC. This result is consistent with the result that the asf1-33 mutant did not require cds1, which encodes a DNA replication checkpoint factor. Binding of Asf1-33 with Histone H3 and Localization of Asf1-33 protein We next examined whether Asf1-33 binds to histone H3 at 36uC. Wild-type Asf1 and Asf1-33 were co-immunoprecipitated with histone H3, but Asf1-33 
did not co-immunoprecipitate with histone H3 at 36uC. The level of histone proteins in the asf1-33 mutant and asf1+ cells was indistinguishable, confirming the mutations of asf1 do not affect histone levels in fission yeast but do lead to alterations in histone H3 binding. We next observed the cellular localization of Asf1-33. Immunofluorescence using an anti-Myc antibody showed mislocalization of Asf1-33-13myc at 36uC. Wild-type Asf1-13myc and Asf1-33-13myc at 26uC were in the nucleus, but at 36uC Asf1-33 was seen throughout the cytoplasm. asf1-33 mutations cause drastic defects in chromatin structure Asf1 is involved in chromatin assembly and disassembly through binding to histones H3/H4. Since the binding of Asf1-33 to histone H3 was impaired, we tested chromatin structure in the asf1-33 mutant using MNase. MNase cuts the linker regions of chromatin DNA, and the digested chromatin DNAs are separated by agarose gel electrophoresis, with the resulting ladder pattern reflecting the chromatin structure. When we performed a MNase assay for the asf1-33 mutant, no significant Asf1 was required for the maintenance of genomic stability The phosphorylation of Chk1 in the asf1-33 mutant suggested that DNA damage occurred in these cells. We therefore tested for DNA double-strand breaks using pulse-field gel electrophoresis.
