White C I, Haber J E
Rosenstiel Basic Medical Sciences Research Center, Waltham, MA 02254.
EMBO J. 1990 Mar;9(3):663-73. doi: 10.1002/j.1460-2075.1990.tb08158.x.
We have identified two novel intermediates of homothallic switching of the yeast mating type gene, from MATa to MAT alpha. Following HO endonuclease cleavage, 5' to 3' exonucleolytic digestion is observed distal to the HO cut, creating a 3'-ended single-stranded tail. This recision is more extensive in a rad52 strain unable to switch. Surprisingly, the proximal side of the HO cut is protected from degradation; this stabilization depends on the presence of the silent copy donor sequences. A second intermediate was identified by a quantitative application of the polymerase chain reaction (PCR). The Y alpha-MAT distal covalent fragment of the switched product appears 30 min prior to the appearance of the MAT proximal Y alpha junction. No covalent joining of MAT distal to HML distal sequences is detected. We suggested that the MAT DNA distal to the HO cut invades the intact donor and is extended by DNA synthesis. This step is prevented in a rad52 strain. These intermediates are consistent with a model for MAT switching in which only the distal side of the HO cut is initially active in strand invasion and transfer of information from the donor.
我们已经鉴定出酵母交配型基因从 MATa 转换为 MATα 的同宗转换的两种新中间体。在 HO 内切核酸酶切割后，在 HO 切割位点的远端观察到 5' 至 3' 的核酸外切酶消化，产生一个 3' 末端的单链尾巴。在无法进行转换的 rad52 菌株中，这种切割更为广泛。令人惊讶的是，HO 切割位点的近端受到保护不被降解；这种稳定化依赖于沉默拷贝供体序列的存在。通过聚合酶链反应（PCR）的定量应用鉴定出了第二种中间体。转换产物的 Yα-MAT 远端共价片段在 MAT 近端 Yα 连接出现前 30 分钟出现。未检测到 MAT 远端与 HML 远端序列的共价连接。我们认为，HO 切割位点远端的 MAT DNA 侵入完整的供体，并通过 DNA 合成进行延伸。这一步骤在 rad52 菌株中被阻止。这些中间体与 MAT 转换模型一致，在该模型中，HO 切割位点的远端最初仅在链侵入和从供体转移信息方面具有活性。
