Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
DNA Repair (Amst). 2010 Jan 2;9(1):58-65. doi: 10.1016/j.dnarep.2009.10.011. Epub 2009 Dec 2.
Bacterial AlkB and three human AlkB homologues (ABH1, ABH2, and ABH3) are Fe(2+)/2-oxoglutarate-dependent oxygenases that directly repair alkylation-damaged DNA. Here, we show that ABH1 unexpectedly has a second activity, cleaving DNA at abasic (AP) sites such as those arising spontaneously from alkylation-dependent depurination reactions. The DNA cleavage activity of ABH1 does not require added Fe(2+) or 2-oxoglutarate, is not inhibited by EDTA, and is unaffected by mutation of the putative metal-binding residues, indicating that this activity arises from an active site distinct from that used for demethylation. AP-specific DNA cleavage was shown to occur by a lyase mechanism, rather than by hydrolysis, with the enzyme remaining associated with the DNA product. ABH1 can cleave at closely spaced AP-sites on opposite DNA strands yielding double-strand breaks in vitro and this reaction may relate to the physiological role of this unexpected AP lyase activity.
细菌 AlkB 及其三种人类同源物（ABH1、ABH2 和 ABH3）是依赖 Fe(2+)/2-氧代戊二酸的加氧酶，可直接修复烷基化损伤的 DNA。在这里，我们发现 ABH1 具有出乎意料的第二种活性，可在碱基缺失（AP）位点切割 DNA，例如那些由依赖烷基化的脱嘌呤反应自发产生的 AP 位点。ABH1 的 DNA 切割活性不需要额外的 Fe(2+)或 2-氧代戊二酸，不受 EDTA 抑制，并且不影响假定的金属结合残基的突变，表明该活性源自与用于去甲基化的活性位点不同的活性位点。AP 特异性 DNA 切割通过裂合酶机制发生，而不是通过水解，酶仍然与 DNA 产物结合。ABH1 可以在相反的 DNA 链上切割紧密间隔的 AP 位点，在体外产生双链断裂，并且该反应可能与这种意外的 AP 裂合酶活性的生理作用有关。
