Negritto M T, Wu X, Kuo T, Chu S, Bailis A M
Department of Molecular Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, California 91010, USA.
Mol Cell Biol. 1997 Jan;17(1):278-86. doi: 10.1128/MCB.17.1.278.
We have developed a system for analyzing recombination between a DNA fragment released in the nucleus from a single-copy plasmid and a genomic target in order to determine the influence of DNA sequence mismatches on the frequency of gene replacement in Saccharomyces cerevisiae. Mismatching was shown to be a potent barrier to efficient gene replacement, but its effect was considerably ameliorated by the presence of DNA sequences that are identical to the genomic target at one end of a chimeric DNA fragment. Disruption of the mismatch repair gene MSH2 greatly reduces but does not eliminate the barrier to recombination between mismatched DNA fragment and genomic target sequences, indicating that the inhibition of gene replacement with mismatched sequences is at least partially under the control of mismatch repair. We also found that mismatched sequences inhibited recombination between a DNA fragment and the genome only when they were close to the edge of the fragment. Together these data indicate that while mismatches can destabilize the relationship between a DNA fragment and a genomic target sequence, they will only do so if they are likely to be in the heteroduplex formed between the recombining molecules.
我们开发了一个系统，用于分析从单拷贝质粒释放到细胞核中的DNA片段与基因组靶点之间的重组，以确定DNA序列错配对酿酒酵母中基因替换频率的影响。结果表明，错配是高效基因替换的一个强大障碍，但嵌合DNA片段一端与基因组靶点相同的DNA序列的存在可显著改善其影响。错配修复基因MSH2的破坏大大降低但并未消除错配DNA片段与基因组靶点序列之间的重组障碍，这表明错配序列对基因替换的抑制至少部分受错配修复的控制。我们还发现，错配序列仅在靠近片段边缘时才会抑制DNA片段与基因组之间的重组。这些数据共同表明，虽然错配会破坏DNA片段与基因组靶点序列之间的关系，但只有当它们可能存在于重组分子之间形成的异源双链体中时才会如此。
