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首页> 外文期刊>Molecular and Cellular Biology >Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation.
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Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation.

机译：双链断裂诱导重组的表征：同源性要求和单链DNA形成。

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In the yeast Saccharomyces cerevisiae, a double-strand chromosome break created by the HO endonuclease is frequently repaired in mitotically growing cells by recombination between flanking homologous regions, producing a deletion. We showed that single-stranded regions were formed on both sides of the double-strand break prior to the formation of the product. The kinetics of the single-stranded DNA were monitored in strains with the recombination-deficient mutations rad52 and rad50 as well as in the wild-type strain. In rad50 mutants, single-stranded DNA was generated at a slower rate than in the wild type, whereas rad52 mutants generated single-stranded DNA at a faster rate. Product formation was largely blocked in the rad52 mutant. In the rad50 rad52 double mutant, the effects were superimposed in that the exonucleolytic activity was slowed but product formation was blocked. rad50 appears to act before or at the same stage as rad52. We constructed strains containing two ura3 segments on one side of the HO cut site and one ura3 region on the other side to characterize how flanking repeats find each other. Deletions formed preterentially between the homologous regions closest to the double-strand break. By varying the size of the middle ura3 segment, we determined that recombination initiated by a double-strand break requires a minimum homologous length between 63 and 89 bp. In these competition experiments, the frequency of recombination was dependent on the length of homology in an approximately linear manner.
机译：在酵母酿酒酵母中，由HO内切核酸酶产生的双链染色体断裂经常通过侧翼同源区域之间的重组在有丝分裂生长的细胞中修复，从而产生缺失。我们表明，在形成产物之前，在双链断裂的两侧均形成了单链区域。在具有重组缺陷突变rad52和rad50的菌株以及野生型菌株中监测单链DNA的动力学。在rad50突变体中，单链DNA的产生速率比在野生型中低，而rad52突变体则以更快的速率产生。在rad52突变体中，产物的形成被大部分阻止。在rad50 rad52双重突变体中，这种作用被叠加，因为放出核酸外切酶的活性减慢了，但是产物的形成被阻止了。 rad50似乎在rad52之前或同时起作用。我们构建的菌株在HO切割位点的一侧包含两个ura3片段，在另一侧包含一个ura3区域，以表征侧翼重复序列如何相互发现。缺失在最接近双链断裂的同源区域之间形成。通过改变中间ura3片段的大小，我们确定了由双链断裂引发的重组需要63至89 bp之间的最小同源长度。在这些竞争实验中，重组频率以近似线性的方式取决于同源性的长度。

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