Frameshift Mutations in Yeast

在基因组复制过程中保持遗传物质的完整性需要非常低的突变率。DNA复制的总体保真度由三个步骤决定：(1)聚合过程中核苷酸掺入的错误率，(2)DNA聚合酶的核外溶核校对活性移除错误碱基配对的末端核苷酸的效率，以及(3)复制后错配修复(MMR)系统移除任何剩余错误的效率。由持久的、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/缺失事件或碱基替换事件。移码突变是指那些不以3的倍数发生的核苷酸插入/缺失事件，从而改变翻译的mRNA的阅读框架。虽然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的功能。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，以及防止固定这种复制错误的编辑功能。该实验室开发了一种移码特异的检测系统，该系统允许在酿酒酵母中很容易地识别出广泛的移码事件。该系统基于LYS2基因座上一个已定义的4个移码等位基因(Lys2deltaBgl)的逆转。先前对野生型、校对缺陷型(Exo-)和MMR缺陷型菌株lys2deltaBgl逆转事件的分析表明，几乎所有的补偿性移码事件都是第二位点，1个碱基的缺失事件。这个项目扩展了以前的分析，有三个具体的目标：1.通过构建在消除复制错误的两个途径上都有缺陷的酵母菌株来扩展先前对lys2deltaBgl逆转事件的分析：核酸外切校对和MMR。对Exo-MMR-株的lys2deltaBgl逆转谱的检查将允许直接确定DNA复制过程中发生的移码错误。Exo-MMR谱与Exo-MMR谱的比较将允许直接确定由MMR机制移除的复制中间体。同样，将Exo-MMR谱与Exo MMR谱进行比较将发现复制错误，有待校对。在校对缺陷(Exo-)突变体中获得的移码光谱将被用于研究酵母DNA聚合酶II和DNA聚合酶III在DNA复制过程中领先与滞后链合成中的相对作用。将在第二号染色体上正常取向的lys2deltaBgl获得的逆转光谱与颠倒该基因座后获得的光谱进行比较，这是一个交换领先和滞后链身份的过程。如果给定的聚合酶专门用于领先或滞后链的合成，那么可以预测，当领先链和滞后链颠倒时，外移码光谱将发生显著变化。相反，如果聚合酶同时参与领先和滞后链的合成，那么当突变靶点被颠倒时，光谱应该不会有明显的变化。Lys2deltaBgl系统允许检查帧移位，但不允许检测另一类主要的帧移位事件：1个BP插入。目前正在开发一种替代lys2deltaBgl系统的系统，该系统将能够识别主要是1个移码事件。这将通过使包含lys2deltaBg1逆转窗口的LYS2区域接受定点突变来实现，这将把-1\f25 FrameShift-1\f6窗口转换为专门识别-1\f25 1-1\f25 1BP-1插入的窗口。与lys2deltaBgl-1移码窗口一样，将检测野生型、MMR缺陷和校对缺陷菌株中1个移码的分子性质。

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.