Cellular Responses to DNA-Protein Crosslinks

环境和内源性接触产生DNA-蛋白质和DNA-多肽交联物的化学物质与几种癌症、哮喘和其他疾病的风险增加相关。目前，美国人口中有成千上万的人接触到了最常见的DNA-蛋白质交联剂之一--甲醛。这些接触发生在职业和家庭环境中，受影响的个人经常经历多年的长期暴露，远远超过典型的室内空气质量标准。虽然目前对这类DNA损伤的细胞修复和反应机制知之甚少，但我们之前的研究既有严格建立的合成化学程序来创建和利用含有定点修饰的DNA-蛋白质交联物的DNA，又有确定剂量依赖的全基因组分析，这些分析确定了其产物具有限制DNA-蛋白质交联物诱导的细胞毒性的基因。这些研究产生了一系列假说，假设真核细胞暴露在长期、低水平的DNA-蛋白质交联剂中，通过同源重组将细胞毒性和突变降至最低，而在急性高剂量暴露后，细胞将转移到由核蛋白酶体依赖的共价连接蛋白降解启动的途径，然后通过核苷酸切除修复或跨损伤DNA聚合酶进行处理。为了实现我们的目标，确定DNA-蛋白质交联链在慢性和急性暴露下修复和耐受的基本途径，基因特异性缺失分析将识别在限制细胞毒性和突变方面至关重要的基因星座和相互关联的途径。单个基因产物在调节细胞对DNA-蛋白质交联物反应中的作用将包括修复、重组、跨损伤合成、细胞周期检查点、染色质重塑和蛋白分解途径。跨损伤合成聚合酶的活性将使用含有位点特异性DNA-肽交联物和随机加成的DPC的DNA来建立。总的来说，这些调查将产生对这类损伤的修复和耐受性的全面分析。

Environmental and endogenous exposure to chemicals that produce DNA-protein and DNA-peptide crosslinks are correlated with an increased risk of several cancers, asthma, and other diseases. Currently many thousands of individuals in the US population are exposed to one of the most common DNA-protein crosslink-inducing agents, formaldehyde. These exposures take place in both occupational and in-home settings and affected individuals often experience multi-year chronic exposures that are far in excess of typical indoor air quality standards. Although little is currently known concerning the cellular repair and response mechanisms for this class of DNA lesions, our prior investigations have both rigorously established synthetic chemical procedures to create and utilize DNAs containing site-specifically modified DNA-protein crosslinks and determined dose-dependent, genome-wide assays that identify genes whose products function to limit DNA-protein crosslink-induced cytotoxicity. These investigations have generated a series of hypotheses which postulate that eukaryotic cells exposed to chronic, low levels of DNA-protein crosslinking agents minimize cytotoxicity and mutagenesis through homologous recombination, while following acute high dose exposure, cells will shift to pathways initiated by nuclear proteasome-dependent degradation of covalently linked proteins that can then be processed via either nucleotide excision repair or translesion DNA polymerases. To accomplish our objective of determining the fundamental pathways for the repair and tolerance of DNA-protein crosslinks under chronic and acute exposures, gene-specific deletion analyses will identify the constellation of genes and interrelated pathways that are critical in limiting cellular toxicity and mutagenesis. The roles of individual gene products in modulating cellular response to DNA-protein crosslinks will include repair, recombination, translesion synthesis, cell cycle check points, chromatin remodeling and proteolytic pathways. The activities of translesion synthesis polymerases will be established using DNAs containing site-specific DNA-peptide crosslinks and randomly adducted DPCs. Collectively, these investigations will yield comprehensive analyses of repair and tolerance of this class of lesions