RNA Polymerase Transcription Past DNA Adducts

描述(申请人提供)：这项研究的长期目标是详细了解DNA中共价修饰的碱基在转录延伸阶段如何影响RNA聚合酶的行为，最终目标是产生详细的结构模型，描绘DNA中发现的大小加合物上的RNA聚合酶相互作用。以下四个具体目标将针对这一点：(1)评估经过特定位点损伤的转录对越过受损或修饰位点的延伸的影响；(2)确定全长转录本的碱基序列，并表征截短转录本3‘端的碱基组成；(3)确定转录过程中DNA加合物上碱基加成的Km和Vrnax值；以及(4)利用计算机模拟技术提供停滞在DNA加合物处的转录复合体的特征。现在很清楚的是，某些DNA损伤可以导致RNA聚合酶在修饰的位置停滞不前，导致转录本被截断，或者越过改变的碱基，产生全长RNA。停滞的转录复合体发出转录偶联DNA修复的信号。转录偶联DNA修复的重要性可以从临床表型异常中观察到，就像在Cockayne综合征患者中所看到的那样，Cockayne综合征是一种以严重生长和发育缺陷为特征的疾病。优先清除来自离散的、活跃的遗传位点的DNA损伤具有广泛和重要的影响：突变中可能存在偏见；当使用特定加合物的优先清除率进行比较时，DNA损伤和修复与肿瘤发生的相关性可能更强；从表达基因的转录链中实际移除加合物可能非常依赖于它们阻碍RNA合成的能力。

DESCRIPTION (provided by applicant): The long range goal of this research is to gain a detailed understanding of how covalently modified bases in DNA affect RNA polymerase behavior during the elongation phase of transcription, with the ultimate objective of producing detailed structural models that portray RNA polymerase interactions at bulky and small adducts found in DNA. The following four Specific Aims will be targeted toward this: (1) to assess the effects of transcription past site-specific lesions on elongation past the damaged or modified site; (2) to determine the base sequence of full-length transcripts, and to characterize the base composition at the 3'- ends of truncated transcripts; (3) to determine Km, and Vrnax values for base addition at DNA adducts during transcription; and (4) to employ computer-modeling techniques to provide characteristics of transcription complexes stalled at DNA adducts. It is now quite clear that certain DNA lesions can cause RNA polymerase to stall at the modified site, resulting in a truncated transcript, or progress past the altered base, producing full-length RNA. Stalled transcription complexes signal transcription-coupled DNA repair. The importance of transcription-coupled DNA repair is evidenced by the clinical phenotype observed when it is aberrant, as is seen in patients with Cockayne's syndrome, a disease characterized by severe growth and developmental defects. There are broad and significant implications for preferential clearance of DNA damage from discrete, active genetic loci: Biases in mutagenesis can exist; correlations of DNA damage and repair with tumorigenesis might be stronger when preferential clearance rates for a particular adduct are used for making the comparisons, rather than total genomic repair; and the actual removal of adducts from the transcribed strand of an expressed gene might be very dependent on their ability to impede RNA synthesis.