FINE-RESOLUTION MAPPING OF SPONTANEOUS AND DOUBLE-STRAND BREAK-INDUCED GENE CONVERSION TRACTS IN SACCHAROMYCES-CEREVISIAE REVEALS REVERSIBLE MITOTIC CONVERSION POLARITY
FINE-RESOLUTION MAPPING OF SPONTANEOUS AND DOUBLE-STRAND BREAK-INDUCED GENE CONVERSION TRACTS IN SACCHAROMYCES-CEREVISIAE REVEALS REVERSIBLE MITOTIC CONVERSION POLARITY
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DOI：
10.1128/mcb.14.6.3863
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发表时间：
1994-06-01
影响因子：
5.3
通讯作者：
NICKOLOFF, JA
中科院分区：
文献类型：
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作者：
SWEETSER, DB;HOUGH, H;NICKOLOFF, JA
自发和双链断裂（DSB）诱导的基因转换进行了研究，在酿酒酵母ura 3基因的等位基因含有9个表型沉默标记和HO核酸酶识别位点。ARS 1/CEN 4质粒上携带的这些等位基因的转换涉及与染色体V上的异等位基因的相互作用，并受到在HO位点产生的DSB的刺激。没有检测到将质粒整合到染色体中的杂交，因为所得的双着丝粒染色体将是致命的。穿梭质粒中转换的等位基因很容易转移到大肠杆菌中，并进行标记转换分析，便于对来自5个杂交的400多个独立产物进行表征。这项分析揭示了基因转换的几个新特征。DSB诱导的转化片段的平均长度为200至300 bp，尽管约20%非常短（小于53 bp）。约20%的自发束也小于53 bp，但自发束平均比DSB诱导的束长约40%。大多数大片是连续的，但3%有不连续的转换模式，这表明广泛的异源双链DNA至少在这一部分的事件中形成。异源双链DNA中的错配在两个方向上被修复，并且观察到短至44 bp的修复区。令人惊讶的是，大多数DSB诱导的基因转换束是单向的，并表现出可逆的极性，这取决于受体和供体等位基因中DSB和移码突变的位置。
Spontaneous and double-strand break (DSB)-induced gene conversion was examined in alleles of the Saccharomyces cerevisiae ura3 gene containing nine phenotypically silent markers and an HO nuclease recognition site. Conversions of these alleles, carried on ARS1/CEN4 plasmids, involved interactions with heteroalleles on chromosome V and were stimulated by DSBs created at HO sites. Crossovers that integrate plasmids into chromosomes were not detected since the resultant dicentric chromosomes would be lethal. Converted alleles in shuttle plasmids were easily transferred to Escherichia coli and analyzed for marker conversion, facilitating the characterization of more than 400 independent products from five crosses. This analysis revealed several new features of gene conversions. The average length of DSB-induced conversion tracts was 200 to 300 bp, although about 20% were very short (less than 53 bp). About 20% of spontaneous tracts also were also less than 53 bp, but spontaneous tracts were on average about 40% longer than DSB-induced tracts. Most tracts were continuous, but 3% had discontinuous conversion patterns, indicating that extensive heteroduplex DNA is formed during at least this fraction of events. Mismatches in heteroduplex DNA were repaired in both directions, and repair tracts as short as 44 bp were observed. Surprisingly, most DSB-induced gene conversion tracts were unidirectional and exhibited a reversible polarity that depended on the locations of DSBs and frameshift mutations in recipient and donor alleles.