Transcription-associated mutagenesis in yeast

酵母中转录相关的诱变

• 批准号: 7873932
• 负责人: SUE JINKS-ROBERTSON
• 金额: $ 30.62万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7873932
• 关键词: Address; Affect; Automobile Driving; Base Excision Repairs; Biological Assay; Biological Models; Bypass; Cell Cycle; Cell Nucleus; Chromosomes; Coupling; Cytosine; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Deamination; Doxycycline; Employee Strikes; Excision; Exhibits; Galactose; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; In Vitro; Left; Link; Malignant Neoplasms; Measures; Methods; Mutagenesis; Mutation; Nucleotide Excision Repair; Nucleotides; Pathway interactions; Pattern; Phase; Process; RNA chemical synthesis; Reaction; Reporter; Reporter Genes; Site; Source; Southern Blotting; Testing; Thymine; Time; Transcription-Coupled Repair; Uracil; Yeasts; base; dUTP pyrophosphatase; design; human disease; insight; novel; overexpression; promoter; public health relevance; repaired; research study; uracil-DNA glycosylase

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.

描述（由申请人提供）：转录对重组和诱变都具有强烈的刺激作用，这些作用与增强的DNA损伤有关。在转录相关诱变的情况下，我们已经发现，脱嘧啶/脱嘌呤（AP）位点在高度转录的酵母DNA中积累到非常高的水平。这些AP位点中的大多数是由尿嘧啶的酶促去除产生的，遗传研究表明，转录相关的尿嘧啶来自直接掺入，而不是来自胞嘧啶脱氨基。尿嘧啶的掺入增强表明，在DNA聚合酶可用的核苷酸库中存在转录相关的扰动，并且所提出的实验旨在进一步研究转录与DNA合成保真度之间的关系。第一个目标将解决高度转录的DNA中的尿嘧啶是否仅来源于dUTP的直接掺入，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掺入到一个未连接的基因，目标2将检查尿嘧啶的掺入是否严格限于高度转录的DNA，或是否可能延伸到相邻区域。尿嘧啶的转录相关掺入可能发生在正常的DNA复制过程中，或者它可能与伴随DNA修复过程的更有限的DNA合成有关。这个问题将在目标3中解决，其中细胞周期调节启动子将用于检查转录相关尿嘧啶掺入是否仅限于S期或是否在整个细胞周期中发生。最后，目的4旨在探索修复途径所表现出的非常独特的“链”的基础，该修复途径去除了在高度转录的DNA中积累的AP位点。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，这种诱变直接与基因表达水平有关，并且可能调节全球突变率和模式。 公共卫生相关性：突变为进化过程提供了原材料，并导致了许多人类疾病，特别是癌症。拟议的实验使用酵母作为模型系统，以探索一种新型的诱变，是专门连接到转录。这些研究将为促进靶向突变积累的机制提供深入了解。