Template switching during break-induced replication is promoted by the Mph1 helicase in Saccharomyces cerevisiae.
Template switching during break-induced replication is promoted by the Mph1 helicase in Saccharomyces cerevisiae.
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DOI：
10.1534/genetics.114.162297
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发表时间：
2014-04
期刊：
影响因子：
3.3
通讯作者：
Symington LS
中科院分区：
文献类型：
--
作者：
Stafa A;Donnianni RA;Timashev LA;Lam AF;Symington LS
只有一个末端与供体双链体同源的染色体双链断裂（DSB）通过链侵入进行修复，然后复制到染色体末端（断裂诱导复制，BIR）。使用基于转化的测定系统，先前表明BIR可以通过几轮链侵入、DNA合成和解离发生。在这里，我们描述了一个修改的转化为基础的测定，以促进检测供体模板之间的切换在BIR期间通过遗传选择在二倍体酵母。除了预期的模板转换产物的恢复，我们发现在BIR期间染色体同源物之间的重组频率很高，这表明DSB从转化线性DNA转移到供体染色体，启动次级重组事件。在mph1Δ突变体中，BIR的频率增加，但模板转换事件的百分比显著降低，揭示了Mph1在促进BIR相关的模板转换中的重要作用。此外，我们表明，Mus81，Rad1，和Yen1结构选择性核酸酶的行为冗余，以促进BIR。
Chromosomal double-strand breaks (DSBs) that have only one end with homology to a donor duplex undergo repair by strand invasion followed by replication to the chromosome terminus (break-induced replication, BIR). Using a transformation-based assay system, it was previously shown that BIR could occur by several rounds of strand invasion, DNA synthesis, and dissociation. Here we describe a modification of the transformation-based assay to facilitate detection of switching between donor templates during BIR by genetic selection in diploid yeast. In addition to the expected recovery of template switch products, we found a high frequency of recombination between chromosome homologs during BIR, suggesting transfer of the DSB from the transforming linear DNA to the donor chromosome, initiating secondary recombination events. The frequency of BIR increased in the mph1Δ mutant, but the percentage of template switch events was significantly decreased, revealing an important role for Mph1 in promoting BIR-associated template switching. In addition, we show that the Mus81, Rad1, and Yen1 structure-selective nucleases act redundantly to facilitate BIR.
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