Cellular responses to DNA-protein crosslinks

描述(由申请人提供)：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癌症、哮喘和其他疾病的风险增加相关。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。最常见的DNA-蛋白质交联剂之一是甲醛，目前美国人口中有数千人接触到这种化合物和其他醛类化合物。为了了解这些DNA损伤的生物过程，我们的实验室已经：i)建立了合成化学程序来创建包含位点特异性修饰的DNA-蛋白质交联物的DNA；ii)确定了能够催化这些损伤的跨损伤合成的DNA聚合酶；以及iii)进行了剂量依赖的全基因组分析，以确定其产物具有限制DNA-蛋白质交联物诱导的细胞毒性的基因。这些研究已经产生了一系列假设，假设当真核细胞暴露在长期、低水平的DNA-蛋白质交联剂中时，细胞毒性和突变可以通过涉及同源重组的耐受途径最小化。然而，在急性高剂量暴露后，假设细胞将转移到涉及核苷酸切除修复或跨损伤合成的途径。为了解决这些假说，将使用基因特异性缺失或siRNA耗尽和小分子抑制剂来确定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可能作用可能包括参与DNA修复、重组、跨损伤合成、细胞周期检查点和蛋白质分解途径。修复中间体的生化分析以及解旋酶和跨损伤合成聚合酶的活性将使用包含特定位点的DNA-肽和蛋白质交联物的DNA来建立。总而言之，这些研究将产生对这类DNA损伤的修复和耐受性的全面分析。

DESCRIPTION (provided by applicant): Environmental and endogenous exposure to chemicals that produce DNA-protein and DNA-peptide crosslinks are correlated with an increased risk of cancer, asthma, and other diseases. 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. One of the most common DNA-protein crosslink-inducing agents is formaldehyde and currently thousands of individuals in the US population are exposed to this and other aldehydic compounds. To understand the biological processing of these DNA lesions, our laboratories have i) established synthetic chemical procedures to create DNAs containing site-specifically modified DNA-protein crosslinks, ii) identified DNA polymerases that are capable of catalyzing translesion synthesis of these lesions, and iii) carried out dose-dependent, genome-wide assays that identified genes whose products function to limit DNA-protein crosslink- induced cytotoxicity. These investigations have generated a series of hypotheses which postulate that when eukaryotic cells are exposed to chronic, low levels of DNA-protein crosslinking agents, cytotoxicity and mutagenesis are minimized by tolerance pathways involving homologous recombination. However, following acute high dose exposures, it is hypothesized that cells will shift to pathways involving components of either nucleotide excision repair or translesion synthesis. To address these hypotheses, gene-specific deletions or siRNA depletion and small molecular inhibitors will be used to identify the constellation of genes and interrelated pathways that are critical in limiting cellular toxicity and mutagenesis. Possible roles of individual gene products in modulating cellular responses to DNA-protein crosslinks may include involvement in DNA repair, recombination, translesion synthesis, cell cycle check points, and proteolytic pathways. Biochemical analyses of repair intermediates and the activities of helicases and translesion synthesis polymerases will be established using DNAs containing site-specific DNA-peptide and protein crosslinks. Collectively, these investigations will yield comprehensive analyses of repair and tolerance of this class of DNA lesions.