Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae. Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3′ overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3′ end affects recombination outcomes and whether the presence of a 3′ versus 5′ overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers versus noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3′ end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3′ versus 5′ overhangs differs, we compared the well-studied 3′ overhang created by I-SceI to a 5′ overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.

中文翻译：

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酵母中不同末端结构的核酸酶产生的有丝分裂双链断裂的重组修复

有丝分裂重组是修复酿酒酵母中双链断裂的主要机制。当前的重组模型主要基于利用酶I- Sce I或HO产生位点特异性断裂的研究，每个断裂均产生具有3'突出端的断裂末端。在这项研究中，使用序列不同的异位底物评估从3'末端频繁去除Polδ介导的错配8个核苷酸是否影响重组结果，以及断裂位点上3'与5'突出端的存在是否发生改变结果。监测的重组结果是重组产物在交叉点与非交叉产物，以及非交叉产物中转移序列（异源双链DNA）的位置/长度。从3'末端开始有22个核苷酸的末端错配很少被清除，与末端的更大距离不会影响重组结果。为了确定带有3'与5'突出端的断裂的重组修复是否不同，我们将经过深入研究的I- Sce I产生的3'突出端与ZFN（锌指核酸酶）产生的5'突出端进行了比较。ZFN的启动产生了更多的重组体，与I- Sce I相比，裂解效率更高，修复率也可能更快。ZFN-中的CO比例高于I- Sce我发起的事件中，两个系统中的NCO在链转移程度方面是无法区分的。这些数据表明，尽管修复率存在潜在差异，但DSB诱导方法和最终极性的最终差异对有丝分裂重组结果影响很小。