Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast.
Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast.
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DOI：
10.1534/g3.120.401603
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发表时间：
2020-10-05
期刊：
影响因子：
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通讯作者：
Jinks-Robertson S
中科院分区：
文献类型：
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作者：
Gamble D;Shaltz S;Jinks-Robertson S
有丝分裂重组是酿酒酵母修复双链断裂的主要机制。目前的重组模型主要基于利用酶I-SceI或HO产生位点特异性断裂的研究，每种断裂都产生具有3′突出端的断裂末端。在这项研究中，使用序列趋异的异位底物来评估频繁的Pol δ介导的从3′端去除8个核苷酸的错配是否会影响重组结果，以及断裂位点处3′与5′突出端的存在是否会改变结果。监测的扩增结果是重组产物在交叉与非交叉中的分布，以及非交叉产物中转移序列（异源双链DNA）的位置/长度。距离3′端22个核苷酸的末端错配很少被去除，距离末端更大的距离不影响重组结果。为了确定具有3′和5′突出端的断裂的重组修复是否不同，我们比较了由I-SceI产生的充分研究的3′突出端和由ZFN（锌指核酸酶）产生的5′突出端。用ZFN起始产生更多的重组体，与相对于I-SceI更有效的切割和潜在更快的修复速率一致。虽然ZFN启动的事件中CO的比例高于I-SceI启动的事件，但两个系统中的NCO在链转移程度方面无法区分。这些数据表明，DSB诱导的方法和由此产生的末端极性差异对有丝分裂重组结果的影响很小，尽管修复率存在潜在差异。
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′ vs. 5′ overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers vs. 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′ vs. 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.