Mizuno K, Emura Y, Baur M, Kohli J, Ohta K, Shibata T
Cellular and Molecular Biology Laboratory, The Institute of Physical and Chemical Research, Wako, Saitama, Japan.
Genes Dev. 1997 Apr 1;11(7):876-86. doi: 10.1101/gad.11.7.876.
The G -->T transversion mutation, ade6-M26, creates the heptanucleotide sequence ATGACTG, which lies close to the 5' end of the open reading frame of the ade6 gene in Schizosaccharomyces pombe. The mutation generates a meiosis-specific recombination hot spot and a binding site for the Mts1/Mts2 protein. We examined the chromatin structure at the ade6 locus in the M26 strain and compared it to that of the wild-type and hot spot-negative control M375. Micrococcal nuclease (MNase) digestion and indirect end-labeling methods were applied. In the M26 strain, we detected a new MNase-hypersensitive site at the position of the M26 mutation and no longer observed the phasing of nucleosomes seen in the wild-type and the M375 strains. Quantitative comparison of MNase sensitivity of the chromatin in premeiotic and meiotic cultures revealed a small meiotic induction of MNase hypersensitivity in the ade6 promoter region of the wild-type and M375 strains. The meiotic induction of MNase hypersensitivity was enhanced significantly in the ade6 promoter region of the M26 strain and also occurred at the M26 mutation site. The formation of the MNase-sensitive region around the heptamer sequence was abolished by the introduction of single-nucleotide substitutions in the heptamer sequence, which also abolish hot spot activity and binding of Mts1/Mts2. These data suggest that Mts1/Mts2 binding to the heptamer sequence results in a chromatin structure suitable for the recruitment of a meiosis-specific recombination function or functions.
G→T颠换突变ade6-M26产生七核苷酸序列ATGACTG,该序列位于粟酒裂殖酵母ade6基因开放阅读框的5'端附近。该突变产生一个减数分裂特异性重组热点和Mts1/Mts2蛋白的结合位点。我们检测了M26菌株中ade6基因座的染色质结构,并将其与野生型和热点阴性对照M375的染色质结构进行了比较。应用了微球菌核酸酶(MNase)消化和间接末端标记方法。在M26菌株中,我们在M26突变位点检测到一个新的MNase高敏位点,并且不再观察到野生型和M375菌株中核小体的相位。对减数分裂前和减数分裂培养物中染色质的MNase敏感性进行定量比较,结果显示野生型和M375菌株的ade6启动子区域存在轻微的减数分裂诱导的MNase高敏性。M26菌株的ade6启动子区域中MNase高敏性的减数分裂诱导显著增强,并且在M26突变位点也出现这种情况。通过在七核苷酸序列中引入单核苷酸取代,消除了七聚体序列周围MNase敏感区域的形成,这也消除了热点活性以及Mts1/Mts2的结合。这些数据表明,Mts1/Mts2与七核苷酸序列的结合导致了一种适合募集一种或多种减数分裂特异性重组功能的染色质结构。