Cho J W, Khalsa G J, Nickoloff J A
Department of Cancer Biology, Harvard University School of Public Health, Boston, MS 02115, USA.
Curr Genet. 1998 Oct;34(4):269-79. doi: 10.1007/s002940050396.
Spontaneous and double-strand break (DSB)-induced gene conversion in Saccharomyces cerevisiae was assayed using non-tandem chromosomal direct repeat crosses and plasmid x chromosome crosses. Each cross involved identical ura3 alleles marked with phenotypically silent restriction fragment length polymorphic (RFLP) mutations at approximately 100-bp intervals. DSBs introduced in vivo at HO sites in one allele stimulated recombination to Ura+ by more than two orders of magnitude. Spontaneous gene-conversion products were isolated from a related strain lacking a functional HO nuclease gene. The multiple markers did not appear to influence the frequency of direct repeat deletions for spontaneous or DSB-induced events. DSB-induced conversion reflected efficient mismatch repair of heteroduplex DNA. Conversion frequencies of equidistant markers on opposites sides of the DSB were similar in the direct repeat cross. In contrast, markers 5' of the DSB (promoter-proximal) converted more often than 3' markers in plasmid x chromosome crosses, a possible consequence of crossing-over associated with long conversion tracts. With direct repeats, bidirectional tracts (extending 5' and 3' of the DSB) occurred twice as often as in a plasmid x chromosome cross in which DSBs were introduced into the plasmid-borne allele. A key difference between the direct-repeat and plasmidxchromosome crosses is that the ends of a broken plasmid are linked, whereas the ends of a broken chromosome are unlinked. We tested whether linkage of ends influenced tract directionality using a second plasmid x chromosome cross in which DSBs were introduced into the chromosomal allele and found few bidirectional tracts. Thus, chromosome environment, but not linkage of ends, influences tract directionality. The similar tract spectra of the two plasmid x chromosome crosses suggest that similar mechanisms are involved whether recombination is initiated by DSBs in plasmid or chromosomal alleles.
利用非串联染色体直接重复杂交和质粒×染色体杂交，对酿酒酵母中的自发和双链断裂（DSB）诱导的基因转换进行了测定。每次杂交都涉及相同的ura3等位基因，这些等位基因在大约100个碱基对的间隔处标记有表型沉默的限制性片段长度多态性（RFLP）突变。在一个等位基因的HO位点体内引入的DSB将重组刺激为Ura +的频率提高了两个多数量级。自发基因转换产物是从缺乏功能性HO核酸酶基因的相关菌株中分离出来的。多个标记似乎不影响自发或DSB诱导事件的直接重复缺失频率。DSB诱导的转换反映了异源双链DNA的有效错配修复。在直接重复杂交中，DSB相对两侧等距标记的转换频率相似。相比之下，在质粒×染色体杂交中，DSB的5'端（启动子近端）比3'端标记转换更频繁，这可能是与长转换片段相关的交叉的结果。对于直接重复，双向片段（从DSB向5'和3'延伸）出现的频率是将DSB引入质粒携带等位基因的质粒×染色体杂交中的两倍。直接重复杂交和质粒×染色体杂交之间的一个关键区别是，断裂质粒的末端是相连的，而断裂染色体的末端是不相连的。我们使用第二次质粒×染色体杂交测试了末端连接是否影响片段方向性，在该杂交中，DSB被引入染色体等位基因，结果发现双向片段很少。因此，染色体环境而非末端连接影响片段方向性。两次质粒×染色体杂交相似的片段谱表明，无论重组是由质粒还是染色体等位基因中的DSB引发，都涉及相似的机制。
