EFFECTS OF DNA LESIONS OF UNTARGETED DNA METABOLIC EVENTS

对DSB的错误修复可能会导致潜在的致命事件，如 基因组重排或缺失。未修复的DSB可能会直接导致 染色体丢失和间接可能是非靶向DNA代谢的介质 事件包括增强重组或复制停滞。我们有 利用位点特异性内切酶HO和MAT开关基因座(YZ 接头)从酿酒酵母中分离出来，研究其分子 性外诱发DSB的后果。45个碱基的YZ结 将带有非同源细菌DNA序列的片段克隆到 可选择的高拷贝2U矢量，并转换为非开关部署 酵母菌ACY522。该菌株随后被第二个菌株转化。 GAL作用下含HO内切酶的可选择载体(PGALHOT) 控制力。YZ交界处DSB切割部位的活体断裂监测 用Southern杂交分析添加半乳糖后的不同时间。戴斯 YZ交界处的破坏最初是在4小时内观察到的，并 在添加半乳糖后10小时达到最大。2U的YZ切割 目标质粒不完整，&gt；50%的超螺旋质粒保持在10 在添加半乳糖后数小时。由于&gt；80%的细胞维持HO 质粒pGALHOT在半乳糖时间过程中，相当一部分 含有双链断裂酶的细胞也含有完整的质粒。电池的电镀 将所选择的Ho和YZ靶质粒的含量降至最低 半乳糖平板导致生长产生严重抑制 细长的丝状细胞的微集落。这些葡萄糖的挽救 微集落表明，这些细胞中的大多数已经死亡。 异步化半乳糖2uYZ和PGALHOT的形态分析 含有细胞的二倍体细胞的数量增加具有特征 辐射诱导的G2细胞周期停滞。细胞学染色 用DAPI或Giemsa染色的双重细胞的细胞核表明 哑铃状形态，细胞核陷于芽峡部。这些 结果与DSB介导的反式细胞停滞是一致的。

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.