Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Genetics. 2012 Feb;190(2):501-10. doi: 10.1534/genetics.111.134890. Epub 2011 Nov 17.
Small insertions or deletions that alter the reading frame of a gene typically occur in simple repeats such as mononucleotide runs and are thought to reflect spontaneous primer-template misalignment during DNA replication. The resulting extrahelical repeat is efficiently recognized by the mismatch repair machinery, which specifically replaces the newly replicated strand to restore the original sequence. Frameshift mutagenesis is most easily studied using reversion assays, and previous studies in Saccharomyces cerevisiae suggested that the length threshold for polymerase slippage in mononucleotide runs is 4N. Because the probability of slippage is strongly correlated with run length, however, it was not clear whether shorter runs were unable to support slippage or whether the resulting frameshifts were obscured by the presence of longer runs. To address this issue, we removed all mononucleotide runs >3N from the yeast lys2ΔBgl and lys2ΔA746 frameshift reversion assays, which detect net 1-bp deletions and insertions, respectively. Analyses demonstrate that 2N and 3N runs can support primer-template misalignment, but there is striking run-specific variation in the frequency of slippage, in the accumulation of +1 vs. -1 frameshifts and in the apparent efficiency of mismatch repair. We suggest that some of this variation reflects the role of flanking sequence in initiating primer-template misalignment and that some reflects replication-independent frameshifts generated by the nonhomologous end-joining pathway. Finally, we demonstrate that nonhomologous end joining is uniquely required for the de novo creation of tandem duplications from noniterated sequence.
小的插入或缺失会改变基因的阅读框架，这种情况通常发生在简单重复序列中，如单核苷酸重复，据认为这反映了 DNA 复制过程中引物-模板的自发错配。由此产生的额外螺旋重复序列被错配修复机制高效识别，该机制特异性地替换新复制的链，以恢复原始序列。移码突变最容易通过回复试验进行研究，先前在酿酒酵母中的研究表明，单核苷酸重复中聚合酶滑动的长度阈值为 4N。然而，由于滑动的概率与重复长度强烈相关，因此不清楚较短的重复序列是否无法支持滑动，或者产生的移码突变是否被较长的重复序列所掩盖。为了解决这个问题，我们从酵母 lys2ΔBgl 和 lys2ΔA746 移码回复试验中去除了所有 >3N 的单核苷酸重复，这两个试验分别检测净 1bp 的缺失和插入。分析表明，2N 和 3N 的重复序列可以支持引物-模板错配，但滑动的频率、+1 与-1 移码的积累以及错配修复的明显效率存在显著的重复特异性变化。我们认为，这种变化的部分原因反映了侧翼序列在引发引物-模板错配中的作用，部分原因反映了非同源末端连接途径产生的与复制无关的移码。最后，我们证明非同源末端连接对于从头从非重复序列创建串联重复是唯一必需的。
