Müller Tina A, Struble Sarah L, Meek Katheryn, Hausinger Robert P
Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA; Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI 48824, USA.
Biochem Biophys Res Commun. 2018 Jan 1;495(1):98-103. doi: 10.1016/j.bbrc.2017.10.158. Epub 2017 Oct 31.
Alkbh1 is a mammalian homolog of the Escherichia coli DNA repair enzyme AlkB, an Fe(II) and 2-oxoglutarate dependent dioxygenase that removes alkyl lesions from DNA bases. The human homolog ALKBH1 has been associated with six different enzymatic activities including DNA, mRNA, or tRNA hydroxylation, cleavage at abasic (AP) sites in DNA, as well as demethylation of histones. The reported cellular roles of this protein reflect the diverse enzymatic activities and include direct DNA repair, tRNA modification, and histone modification. We demonstrate that ALKBH1 produced in mammalian cells (ALKBH1) is similar to the protein produced in bacteria (ALKBH1) with regard to its mA demethylase and AP lyase activities. In addition, we find that ALKBH1 forms a covalent adduct with the 5' product of the lyase product in a manner analogous to ALKBH1. Localization and subcellular fractionation studies with the endogenous protein in two human cell strains confirm that ALKBH1 is primarily in the mitochondria. Two strains of CRISPR/Cas9-created ALKBH1-deficient HEK293 cells showed increases in mtDNA copy number and mitochondrial dysfunction as revealed by growth measurements and citrate synthase activity assays.
Alkbh1是大肠杆菌DNA修复酶AlkB的哺乳动物同源物，AlkB是一种依赖于Fe(II)和2-氧代戊二酸的双加氧酶，可从DNA碱基上去除烷基损伤。人类同源物ALKBH1与六种不同的酶活性相关，包括DNA、mRNA或tRNA羟基化、DNA中无碱基（AP）位点的切割以及组蛋白的去甲基化。该蛋白已报道的细胞作用反映了其多样的酶活性，包括直接DNA修复、tRNA修饰和组蛋白修饰。我们证明，在哺乳动物细胞中产生的ALKBH1（ALKBH1）在其mA去甲基酶和AP裂解酶活性方面与在细菌中产生的蛋白（ALKBH1）相似。此外，我们发现ALKBH1以类似于ALKBH1的方式与裂解酶产物的5'产物形成共价加合物。对两种人类细胞系中内源性蛋白的定位和亚细胞分级分离研究证实，ALKBH1主要存在于线粒体中。通过生长测量和柠檬酸合酶活性测定发现，两株经CRISPR/Cas9技术构建的ALKBH1缺陷型HEK293细胞的线粒体DNA拷贝数增加且线粒体功能障碍。
