Mechanisms for Transcription-Coupled Repair in Human Cells

DNA修复缺陷性遗传病会导致明显的癌症易感性，许多还会导致发育、神经或免疫异常，和/或早衰。在避免癌变和正常发育过程中，DNA修复的隐含要求被认为与细胞代谢产生的活性氧以及电离辐射等环境因素造成的DNA损伤有关。本项目的主要目标是阐明转录偶联修复（TCR）的分子机制和人类细胞氧化DNA损伤的处理过程，以便更好地了解这些过程中遗传缺陷对人类健康的影响。该方法涉及对高度多效性的人类DNA修复蛋白XPG的多个关键功能的综合表征，XPG在多个DNA修复过程中起整合作用。XPG的酶活性在核苷酸切除修复（NER）中是严格要求的，因为它在切除紫外线和大块致癌物损伤时做了第一个切口。这种功能的缺陷导致了高度易发癌变的色素性干皮病（XP）。XPG在氧化DNA损伤的碱基切除修复（BER）中也有重要的非酶作用，通过协调和刺激损伤去除的早期步骤，以及在转录偶联修复（TCR）中，通过与停顿的RNA聚合酶和其他TCR蛋白相互作用，对活性基因转录链的损伤进行优先修复。XPG的突变使这些非酶功能失活，导致严重的发育和神经障碍科凯恩综合征（CS）。需要验证的假设是：(a) XPG和CSB在TCR的早期阶段起着关键作用，这对于对环境DNA损伤的反应和正常生长条件下基因组功能的维持都很重要；(b) XPG在氧化损伤的整体BER中也起着重要的生物学作用。假设这两种功能在预防辐射和化学诱导的致癌作用中都很重要，包括氧化DNA损伤以及正常的产后发育。研究人员建议(1)确定XPG和CSB在氧化DNA损伤修复中的作用，并确定是否涉及全局或转录偶联机制，或两者兼有；(2)研究细胞中TCR复合物的组装对DNA氧化损伤的响应；(3)研究TCR蛋白CSB的CS相关突变片段及其与XPG的相互作用对细胞氧化DNA损伤反应和CS表型的影响；(4)确定TCR的早期步骤，并研究TCR蛋白对停滞的RNA聚合酶II进行重塑以实现修复。这些研究解决了在细胞和环境氧化DNA损伤面前维持基因组完整性和功能的关键机制

DNA repair-defective genetic diseases result in marked cancer predisposition, and many also produce developmental, neurological, or immunological abnormalities, and/or premature aging. The implied requirements for DNA repair in avoidance of carcinogenesis and in normal development are proposed to be related to DNA damage from reactive oxygen species generated by the cellular metabolism as well as by environmental agents including ionizing radiation. The broad objective of this project is to elucidate both the molecular mechanisms for transcription-coupled repair (TCR) and the processing of oxidative DNA damage in human cells, in order to better understand the human health effects of genetic defects in these processes. The approach involves comprehensive characterization of the multiple critical functions of the highly pleiotropic human DNA repair protein XPG, which plays an integrating role in multiple DNA repair processes. The enzymatic activity of XPG is strictly required in nucleotide excision repair (NER) for making the first incision in removal of UV and bulky carcinogen damage. Defects in this function result in the highly cancer- prone disease xeroderma pigmentosum (XP). XPG also has important non-enzymatic roles in base excision repair (BER) of oxidative DNA damage through coordination and stimulation of early steps in lesion removal and in transcription-coupled repair (TCR) - the preferential repair of lesions oh transcribed strands of active genes - through interaction with stalled RNA polymerase and other TCR proteins. Mutations in XPG that inactivate these non-enzymatic functions result in the profound developmental and neurological disorder Cockayne syndrome (CS). The hypotheses to be tested are that (a) XPG together with CSB has a critical role in the early steps of TCR that is important both for responses to environmental DNA damage and for maintenance of genome function under normal growth conditions, and that (b) XPG additionally has biologically important roles in global BER of oxidative damage. Both functions are hypothesized to be important in prevention of radiation- and chemical-induced carcinogenesis involving oxidative DNA damage as well as for normal postnatal development. It is proposed (1) to characterize the roles of XPG and CSB in repair of oxidative DNA damage and determine whether global or transcription-coupled mechanisms, or both, are involved; (2) to investigate the assembly of TCR complexes in cells in response to oxidative DNA damage; (3) to investigate the effect of a CS-related mutant fragment of the TCR protein CSB and its interaction with XPG on cellular responses to oxidative DNA damage and the CS phenotype; and (4) to define early steps in TCR and investigate a proposed remodeling of stalled RNA polymerase II by TCR proteins to enable repair. These studies address key mechanisms for maintaining genomic integrity and function in the face of cellular and environmental oxidative DNA damage