Nelson H H, Sweetser D B, Nickoloff J A
Department of Cancer Biology, Harvard University School of Public Health, Boston, Massachusetts, USA.
Mol Cell Biol. 1996 Jun;16(6):2951-7. doi: 10.1128/MCB.16.6.2951.
Double-strand breaks (DSBs) greatly enhance gene conversion in the yeast Saccharomyces cerevisiae. In prior plasmid x chromosome crosses, conversion tracts were often short ( < 53 bp) and usually extended in only one direction from a DSB in an HO recognition sequence inserted into ura3. To allow fine-structure analysis of short and unidirectional tracts, phenotypically silent markers were introduced at 3- and 6-bp intervals flanking the HO site. These markers, which created a 70-bp homeologous region (71% homology), greatly increased the proportion of bidirectional tracts. Among products with short or unidirectional tracts, 85% were highly directional, converting markers on only one side (the nearest marker being 6 bp from the HO site). A DSB in an HO site insertion creates terminal nonhomologies. The high degree of directionality is a likely consequence of the precise cleavage at homology/nonhomology borders in hybrid DNA by Rad1/10 endonuclease. In contrast, terminal homeology alone yielded mostly unidirectional tracts. Thus, nonhomology flanked by homeology yields primarily bidirectional tracts, but terminal homeology or nonhomology alone yields primarily unidirectional tracts. These results are inconsistent with uni- and bidirectional tracts arising from one- and two-ended invasion mechanisms, respectively, as reduced homology would be expected to favor one-ended events. Tract spectra with terminal homeology alone with similar in RAD1 and rad1 cells, indicating that the high proportion of bidirectional tracts seen with homeology flanking nonhomology is not a consequence of Rad1/10 cleavage at homology/homeology boundaries. Instead, tract directionality appears to reflect the influence of the degree of broken-end homology on mismatch repair.
双链断裂（DSB）极大地增强了酿酒酵母中的基因转换。在先前的质粒与染色体交叉实验中，转换片段通常较短（<53 bp），并且通常仅从插入ura3的HO识别序列中的DSB向一个方向延伸。为了对短的单向片段进行精细结构分析，在HO位点两侧以3 bp和6 bp的间隔引入了表型沉默标记。这些标记创建了一个70 bp的同源区域（71%同源性），大大增加了双向片段的比例。在具有短片段或单向片段的产物中，85%是高度定向的，仅转换一侧的标记（最近的标记距离HO位点6 bp）。HO位点插入中的DSB会产生末端非同源性。高度的方向性可能是Rad1/10核酸内切酶在杂交DNA的同源性/非同源性边界处精确切割的结果。相比之下，仅末端同源性主要产生单向片段。因此，同源性侧翼的非同源性主要产生双向片段，但仅末端同源性或非同源性主要产生单向片段。这些结果与分别由单端和双端侵入机制产生的单向和双向片段不一致，因为预期降低的同源性会有利于单端事件。仅具有末端同源性的片段谱在RAD1和rad1细胞中相似，表明在非同源性侧翼具有同源性时看到的高比例双向片段不是Rad1/10在同源性/同源性边界处切割的结果。相反，片段方向性似乎反映了断裂末端同源程度对错配修复的影响。
