Department of Biology, St. Michael's College, Colchester, VT 05439, USA.
Proc Natl Acad Sci U S A. 2011 Jan 11;108(2):698-703. doi: 10.1073/pnas.1012363108. Epub 2010 Dec 21.
High levels of transcription in Saccharomyces cerevisiae are associated with increased genetic instability, which has been linked to DNA damage. Here, we describe a pGAL-CAN1 forward mutation assay for studying transcription-associated mutagenesis (TAM) in yeast. In a wild-type background with no alterations in DNA repair capacity, ≈50% of forward mutations that arise in the CAN1 gene under high-transcription conditions are deletions of 2-5 bp. Furthermore, the deletions characteristic of TAM localize to discrete hotspots that coincide with 2-4 copies of a tandem repeat. Although the signature deletions of TAM are not affected by the loss of error-free or error-prone lesion bypass pathways, they are completely eliminated by deletion of the TOP1 gene, which encodes the yeast type IB topoisomerase. Hotspots can be transposed into the context of a frameshift reversion assay, which is sensitive enough to detect Top1-dependent deletions even in the absence of high transcription. We suggest that the accumulation of Top1 cleavage complexes is related to the level of transcription and that their removal leads to the signature deletions. Given the high degree of conservation between DNA metabolic processes, the links established here among transcription, Top1, and mutagenesis are likely to extend beyond the yeast system.
在酿酒酵母中，高水平的转录与遗传不稳定性增加有关，而遗传不稳定性又与 DNA 损伤有关。在这里，我们描述了一种 pGAL-CAN1 正向突变测定法，用于研究酵母中转录相关诱变（TAM）。在不改变 DNA 修复能力的野生型背景下，在高转录条件下，CAN1 基因中出现的约 50%的正向突变是 2-5bp 的缺失。此外，TAM 的特征缺失定位于离散的热点，与串联重复的 2-4 个拷贝重合。尽管 TAM 的特征缺失不受无差错或易错损伤旁路途径的丧失影响，但它们完全被编码酵母 I 型拓扑异构酶的 TOP1 基因缺失所消除。热点可以转位到移框回复测定的背景中，即使在没有高转录的情况下，该测定也足够灵敏，足以检测到依赖 Top1 的缺失。我们认为，Top1 切割复合物的积累与转录水平有关，而它们的去除导致了特征缺失。鉴于 DNA 代谢过程之间具有高度的保守性，这里建立的转录、Top1 和诱变之间的联系很可能超出酵母系统。
