Transcription-associated mutagenesis in yeast

描述（由申请人提供）：转录可以对重组和诱变产生强烈的刺激作用，这种作用与增强的DNA损伤有关。在转录相关突变的情况下，我们发现ap嘧啶/apurinic （AP）位点在高度转录的酵母DNA中积累到非常高的水平。大多数AP位点是由酶解尿嘧啶产生的，遗传学研究表明，与转录相关的尿嘧啶是直接结合而不是胞嘧啶脱氨产生的。尿嘧啶的掺入增强表明，DNA聚合酶可用的核苷酸池中存在转录相关的扰动，并且拟议的实验旨在进一步研究转录与DNA合成保真度之间的关系。第一个目的是研究高转录DNA中的尿嘧啶是否仅仅来源于dUTP的直接结合，或者RNA合成中使用的UTP是否也可以在DNA合成过程中取代dTTP。虽然遗传数据表明，报告基因的高转录不会提高尿嘧啶与非连锁基因的结合，但Aim 2将研究尿嘧啶的增强结合是否严格限于高转录的DNA，或者它是否可能延伸到邻近区域。转录相关的尿嘧啶掺入可能发生在正常的DNA复制过程中，也可能与DNA修复过程中更有限的DNA合成有关。这个问题将在Aim 3中得到解决，其中细胞周期调控启动子将用于检查转录相关的尿嘧啶掺入是否仅限于S期或是否发生在整个细胞周期中。最后，Aim 4旨在探索修复途径所表现出的非常独特的“链结性”的基础，这些修复途径去除高度转录DNA中积累的AP位点。总之，这些实验将极大地增强我们对一种非常新颖的诱变来源的理解，这种来源与基因表达水平直接相关，并可能调节全球突变率和模式。

DESCRIPTION (provided by applicant): Transcription can have a strong stimulatory effect on both recombination and mutagenesis, effects that have been linked to enhanced DNA damage. In the case of transcription-associated mutagenesis, we have found that apyrimidinic/apurinic (AP) sites accumulate to a very high level in highly-transcribed yeast DNA. Most of these AP sites are generated by the enzymatic removal of uracil, and genetic studies demonstrate that the transcription-associated uracil is derived from direct incorporation rather than from cytosine deamination. The enhanced incorporation of uracil suggests that there is a transcription-associated perturbation in the nucleotide pool available to DNA polymerases, and the proposed experiments are designed to further examine the relationship between transcription and the fidelity of DNA synthesis. The first aim will address whether the uracil in highly-transcribed DNA is derived solely from the direct incorporation of dUTP, or whether the UTP used in RNA synthesis can also replace dTTP during DNA synthesis. While genetic data suggest that high transcription of a reporter gene does not elevate uracil incorporation into an unlinked gene, Aim 2 will examine whether the enhanced incorporation of uracil is strictly limited to highly-transcribed DNA, or whether it might extend into adjacent regions. The transcription-associated incorporation of uracil could occur during normal DNA replication, or it could specifically be associated with the more limited DNA synthesis that accompanies DNA repair processes. This issue will be addressed in Aim 3, where cell-cycle regulated promoters will be used to examine whether transcription-associated uracil incorporation is limited to S phase or whether it occurs throughout the cell cycle. Finally, Aim 4 is designed to explore the basis of the very distinctive "strandedness" exhibited by the repair pathways that remove the AP sites that accumulate in highly-transcribed DNA. Together these experiments will greatly enhance our understanding of a very novel source of mutagenesis that is directly linked to the level of gene expression, and that likely modulates global mutation rates and patterns. PUBLIC HEALTH RELEVANCE: Mutations provide the raw material for evolutionary processes and are causative in a number of human diseases, especially cancer. The proposed experiments use yeast as a model system to explore a novel type of mutagenesis that is specifically linked to transcription. These studies will provide insight into mechanisms that promote targeted mutation accumulation.