EFFECTS OF DNA LESIONS OF UNTARGETED DNA METABOLIC EVENTS

DSB的不准确修复可能导致潜在的致命事件， 基因组重排或缺失。未修复的DSB可直接导致 染色体丢失和间接可能介导非靶向DNA代谢 包括增强的重组或复制停滞的事件。我们有 利用位点特异性核酸内切酶HO和MAT转换基因座（YZ junction）从酵母S.酿酒酵母来研究 染色体外诱导的DSB的后果。45 bp YZ连接 将侧翼为非同源细菌DNA序列的片段克隆到 可选择的高拷贝2u载体并转化为非转换的deploy 酵母菌株ACY 522。随后用第二个菌株转化该菌株 可选择质粒（pGALHOT），其含有GAL下的HO内切核酸酶 控制监测YZ连接DSB切割部位的体内断裂， 在半乳糖添加后的不同时间，使用Southern印迹分析。DS 在4小时时最初观察到YZ连接处的断裂， 在加入半乳糖后10小时最大。2u的YZ切割 靶质粒不完整，在10 ℃下剩余> 50%的超螺旋质粒 半乳糖添加后10小时。由于> 80%的细胞维持HO 质粒pGALHOT在半乳糖时程中， 具有DSB的细胞也含有未断裂的质粒。细胞平板接种 含有选择下的HO和YZ靶质粒， 半乳糖平板导致严重的生长抑制， 细长丝状细胞的小菌落。葡萄糖拯救这些 小菌落表明这些细胞中的大多数已经死亡。 非同步化半乳糖生长的2uYZ和PGALHOT的形态分析 含有细胞的细胞显示出增加的双重细胞特征， 辐射导致的G2期细胞周期阻滞细胞学染色 使用DAPI或Giemsa染色的双联体细胞的细胞核表明， 哑铃形，细胞核陷在芽峡部。这些 结果与DSB介导的反式细胞停滞一致。

Inaccurate repair of DSBs can lead to such potentially lethal events as genomic rearrangements or deletion. Unrepaired DSBs can lead directly to chromosome loss and indirectly may be mediators of untargeted DNA metabolic events including enhanced recombination or replication arrest. We have utilized the site specific endonuclease HO and the MAT switching locus (YZ junction) from the yeast S. cerevisiae to investigate the molecular consequences of extrachromasomally induced DSBs. A 45 bp YZ junction fragment flanked by nonhomologous bacterial DNA sequences was cloned into a selectable high copy 2u vector and transformed into a nonswitching deploid yeast strain ACY522. This strain was subsequently transformed with a second selectable plasmid (pGALHOT) containing the HO endonuclease under GAL control. In vivo breakage of the YZ junction DSB cut site was monitored at various times following galactose addition using Southern blot analysis. DS breakage at the YZ junction was initially observed at 4 hours and was maximal at 10 hours following galactose addition. YZ cutting of the 2u target plasmid was incomplete with >50% supercoiled plasmid remaining at 10 hours after galactose addition. Since >80% of the cells maintain the HO plasmid pGALHOT during the galactose time course, a significant fraction of the cells with DSBs also contain unbroken plasmid. Plating of cells containing the HO and YZ target plasmids under selection to minimal galactose plates results in a severe inhibition of growth producing microcolonies of elongated filamentous cells. Glucose rescue of these microcolonies suggest that the majority of these cells are dead. Morphological analysis of unsynchronized galactose grown 2uYZ and PGALHOT containing cells show an increased number of doublet cells characteristic of a radiation induced cell cycle blockage in G2. Cytological staining of cell nuclei of doublet cells using DAPI or giemsa stains indicate a dumbbell morphology with the nucleus trapped in the bud isthmus. These results are consistent with DSBs mediating cellular arrest in trans.