Department of Immunology, 1959 NE Pacific St. Seattle, 98195 WA, United States.
Department of Immunology, 1959 NE Pacific St. Seattle, 98195 WA, United States; Department of Biochemistry, University of Washington Medical School, 1959 NE Pacific St. Seattle, 98195 WA, United States of America.
DNA Repair (Amst). 2020 May;89:102825. doi: 10.1016/j.dnarep.2020.102825. Epub 2020 Feb 19.
Proteins form adducts with nucleic acids in a variety of contexts, and these adducts may be cytotoxic if not repaired. Here we apply a proteomic approach to identification of proteins adducted to DNA or RNA in normally proliferating cells. This approach combines RADAR fractionation of proteins covalently bound to nucleic acids with quantitative mass spectrometry (MS). We demonstrate that "RADAR-MS" can quantify induction of TOP1- or TOP2-DNA adducts in cells treated with topotecan or etoposide, respectively, and also identify intermediates in physiological adduct repair. We validate RADAR-MS for discovery of previously unknown adducts by determining the repertoires of adducted proteins in two different normally proliferating human cell lines, CCRF-CEM T cells and GM639 fibroblasts. These repertoires are significantly similar with one another and exhibit robust correlations in their quantitative profiles (Spearman r = 0.52). A very similar repertoire is identified by the classical approach of CsCl buoyant density gradient centrifugation. We find that in normally proliferating human cells, the repertoire of adducted proteins - the "adductome" - is comprised of a limited number of proteins belonging to specific functional groups, and that it is greatly enriched for histones, HMG proteins and proteins involved in RNA splicing. Treatment with low concentrations of formaldehyde caused little change in the composition of the repertoire of adducted proteins, suggesting that reactive aldehydes generated by ongoing metabolic processes may contribute to protein adduction in normally proliferating cells. The identification of an endogenous adductome highlights the importance of adduct repair in maintaining genomic structure and the potential for deficiencies in adduct repair to contribute to cancer.
蛋白质在各种情况下与核酸形成加合物，如果这些加合物得不到修复，可能具有细胞毒性。在这里，我们应用蛋白质组学方法来鉴定正常增殖细胞中与 DNA 或 RNA 结合的蛋白质。这种方法结合了 RADAR 对共价结合核酸的蛋白质的分级分离和定量质谱 (MS)。我们证明，“RADAR-MS”可以定量检测拓扑异构酶 I 或拓扑异构酶 II 与 DNA 的加合物在分别用拓扑替康或依托泊苷处理的细胞中的诱导情况，并且还可以鉴定生理修复过程中的中间产物。我们通过确定两种不同的正常增殖的人细胞系（CCRF-CEM T 细胞和 GM639 成纤维细胞）中加合物修饰蛋白的谱，验证了 RADAR-MS 用于发现先前未知加合物的能力。这些谱彼此非常相似，其定量特征具有很强的相关性（Spearman r=0.52）。通过经典的 CsCl 等密度梯度离心方法也可以鉴定到非常相似的谱。我们发现，在正常增殖的人类细胞中，加合物修饰蛋白的谱（“加合物组”）由属于特定功能组的有限数量的蛋白质组成，并且富含组蛋白、HMG 蛋白和参与 RNA 剪接的蛋白质。用低浓度的甲醛处理对加合物修饰蛋白谱的组成几乎没有影响，这表明代谢过程中产生的反应性醛可能会导致正常增殖细胞中的蛋白质加合物形成。内源性加合物组的鉴定突出了修复在维持基因组结构中的重要性，以及修复不足可能导致癌症的潜在风险。
