Frameshift Mutations in Yeast

在基因组复制期间保持遗传物质的完整性要求突变率非常低。DNA复制的总体保真度由三个步骤确定：（1）聚合过程中核苷酸掺入的错误率，（2）DNA聚合酶的核酸外切酶校正活性去除不正确碱基配对的末端核苷酸的效率，以及（3）复制后错配修复（MMR）系统去除任何剩余错误的效率。由持续的、未修复的复制错误引起的突变通常可以被分类为涉及少量核苷酸的插入/缺失事件或碱基取代事件。移码突变具体地说是那些核苷酸插入/缺失事件，其不以三的倍数发生，并因此改变翻译的mRNA的阅读框架。尽管碱基替换事件通常是表型沉默的，但移码突变几乎总是损害蛋白质功能。鉴于移码突变的非常有害的性质，重要的是要了解在DNA复制过程中产生插入/缺失的机制，以及防止这种复制错误固定的编辑功能。该实验室开发了一种移码特异性检测系统，可以在酵母Saccharomyces cerevisiae中轻松识别广谱移码事件。该系统基于在LYS 2基因座处的确定的+4移码等位基因（lys 2deltaBgl）的回复。先前对野生型、校对缺陷型（Exo-）和MMR缺陷型菌株中的lys 2deltaBgl回复事件的分析已经证明，几乎所有的补偿性移码都是第二位点，1bp缺失事件。本项目是对以往分析的拓展，有三个具体目标：1.将通过构建在去除复制错误的两种途径（核酸外切校正和MMR）中都有缺陷的酵母菌株来扩展对lys 2deltaBgl回复事件的先前分析。在Exo-MMR-菌株中检查lys 2deltaBgl回复谱将允许直接确定在DNA复制期间产生的移码错误。Exo-MMR-光谱与Exo-MMR+光谱的比较将允许直接测定由MMR机制去除的复制中间体。类似地，Exo-MMR-谱与Exo+MMR-谱的比较将揭示复制错误，这需要校正。在校对缺陷型（Exo-）突变体中获得的移码谱将用于研究酵母DNA聚合酶II和DNA聚合酶III在DNA复制过程中前导链合成与滞后链合成的相对作用。将在染色体II上以其正常方向用lys 2deltaBgl获得的回复光谱与反转基因座后获得的光谱进行比较，反转基因座是一个转换前导链和滞后链的身份的过程。如果给定的聚合酶专门用于前导链或滞后链合成，则可以预测，在前导链和滞后链逆转后，外移码谱应该显著变化。相反，如果聚合酶同时参与前导链和滞后链的合成，那么当突变靶标被反转时，谱不应发生显著变化。lys 2deltaBgl系统允许检查-1移码，但不允许检测其他主要类型的移码事件：1bp插入。正在开发一种替代lys 2deltaBgl系统，其将允许鉴定占主导地位的+1移码事件。这将通过对含有lys 2deltaBgl回复窗口的LYS 2区域进行定点诱变来实现，其将-1移码窗口转化为用于特异性鉴定Ibp插入的窗口。与lys 2deltaBgl-1移码窗口一样，将检查野生型、MMR缺陷型和校对缺陷型菌株中+1移码的分子性质。

Maintenance of the integrity of genetic material during genome duplication requires that mutation rates be very low. The overall fidelity of DNA replication is determined at three steps: (1) the error rate of nucleotide incorporation during polymerization, (2) the efficiency with which the exonucleolytic proofreading activity of DNA polymerase removes terminal nucleotides that are incorrectly base-paired, and (3) the efficiency with which post-replicative mismatch repair (MMR) systems remove any remaining errors. Mutations that result from persistent, unrepaired replication errors generally can be classified as either insertion/deletion events involving a small number of nucleotides or base substitution events. Frameshift mutations are specifically those nucleotide insertion/deletion events that do not occur in multiples of three and consequently alter the reading frame of the translated mRNA. Whereas base substitution events often are phenotypically silent, frameshift mutations almost invariably compromise protein function. Given the very deleterious nature of frameshift mutations, it is important to understand the mechanisms for generating insertions/deletions during DNA replication, as well as the editing functions that prevent fixation of such replication errors. This laboratory has developed a frameshift-specific assay system that allows a broad spectrum of frameshift events to be readily identified in the yeast Saccharomyces cerevisiae. This system is based on reversion of a defined +4 frameshift allele (lys2deltaBgl) at the LYS2 locus. Previous analyses of lys2deltaBgl reversion events in wild type, proofreading-defective (Exo-) and MMR-defective strains have demonstrated that almost all of the compensatory frameshifts are second site, 1 bp deletion events. This project extends the previous analyses and has three specific aims:1. Previous analyses of lys2deltaBgl reversion events will be extended by constructing yeast strains that are defective in both of the pathways that remove replication errors: exonucleolytic proofreading and MMR. An examination of the lys2deltaBgl reversion spectrum in Exo-MMR- strains will allow a direct determination of the frameshift errors that are made during DNA replication. Comparison of the Exo-MMR-spectrum to the Exo-MMR+ spectrum will allow direct determination of the replication intermediates removed by the MMR machinery. Similarly, comparison of the Exo-MMR- spectrum to the Exo+MMR- spectrum will reveal replication errors subject to proofreading.2. Frameshift spectra obtained in proofreading-defective (Exo-) mutants will be used to investigate the relative roles of yeast DNA polymerase II and DNA polymerase III in leading versus lagging strand synthesis during DNA replication. Reversion spectra obtained with lys2deltaBgl in its normal orientation on chromosome II will be compared with spectra obtained after inverting the locus, a process which switches the identities of the leading and lagging strands. If a given polymerase is specialized for either leading or lagging strand synthesis, then one would predict that the Exo- frameshift spectrum should change significantly upon reversal of the leading and lagging strands. In contrast, if a polymerase is involved in both leading and lagging strand synthesis, then the spectrum should not change significantly when the mutational target is inverted.3. The lys2deltaBgl system allows examination of -1 frameshifts, but does not allow the detection of the other major class of frameshift events: 1 bp insertions. An alternative to the lys2deltaBgl system that will allow identification of predominantly + 1 frameshift events is being developed. This will be accomplished by subjecting the region of LYS2 containing the lys2deltaBgl reversion window to site-directed mutagenesis, which will convert the -1 frameshift window into a window for specifically identifying 1 bp insertions. As with the lys2deltaBgl -1 frameshift window, the molecular natures of +1 frameshifts in wild type, MMR-defective and proofreading-defective strains will be examined.