Copper Inhibits the AlkB Family DNA Repair Enzymes under Wilson's Disease Condition.

Disturbed metabolism of copper ions can cause diseases such as Wilson's disease (WD). In this work, we investigated the inhibitory effect of Cu(II) ion in vitro on the AlkB family DNA repair enzymes, which are members of the Fe(II)/alpha-ketoglutarate-dependent dioxygenase and include human ALKBH2, ALKBH3, and E. coli AlkB proteins. None of the three proteins was significantly inhibited under normal cellular copper concentrations. However, under WD related condition, we observed that the activities of all three enzymes were strongly suppressed (from 95.2 to 100.0%). We also noted the repair efficiency under ds-DNA condition was less susceptible than ss-DNA to the inhibition.

Oncometabolites d- and l-2-Hydroxyglutarate Inhibit the AlkB Family DNA Repair Enzymes under Physiological Conditions.

Cancer-associated mutations often lead to perturbed cellular energy metabolism and accumulation of potentially harmful oncometabolites. One example is the chiral molecule 2-hydroxyglutarate (2HG); its two stereoisomers (d- and l-2HG) have been found at abnormally high concentrations in tumors featuring anomalous metabolic pathways. 2HG has been demonstrated to competitively inhibit several α-ketoglutarate (αKG)- and non-heme iron-dependent dioxygenases, including some of the AlkB family DNA repair enzymes, such as ALKBH2 and ALKBH3. However, previous studies have only provided the IC50 values of d-2HG on the enzymes, and the results have not been correlated to physiologically relevant concentrations of 2HG and αKG in cancer cells. In this work, we performed detailed kinetic analyses of DNA repair reactions catalyzed by ALKBH2, ALKBH3, and the bacterial AlkB in the presence of d- and l-2HG in both double- and single-stranded DNA contexts. We determined the kinetic parameters of inhibition, including kcat, KM, and Ki. We also correlated the relative concentrations of 2HG and αKG previously measured in tumor cells with the inhibitory effect of 2HG on the AlkB family enzymes. Both d- and l-2HG significantly inhibited the human DNA repair enzymes ALKBH2 and ALKBH3 at pathologically relevant concentrations (73-88% for d-2HG and 31-58% for l-2HG inhibition). This work provides a new perspective that the elevation of the d- or l-2HG concentration in cancer cells may contribute to an increased mutation rate by inhibiting the DNA repair performed by the AlkB family enzymes and thus exacerbate the genesis and progression of tumors.

Adaptive Response Enzyme AlkB Preferentially Repairs 1-Methylguanine and 3-Methylthymine Adducts in Double-Stranded DNA.

The AlkB protein is a repair enzyme that uses an α-ketoglutarate/Fe(II)-dependent mechanism to repair alkyl DNA adducts. AlkB has been reported to repair highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in ss-DNA than in ds-DNA. Here, we tested the repair of weaker AlkB substrates 1-methylguanine and 3-methylthymine and found that AlkB prefers to repair them in ds-DNA. We also discovered that AlkB and its human homologues, ABH2 and ABH3, are able to repair the aforementioned adducts when the adduct is present in a mismatched base pair. These observations demonstrate the strong adaptability of AlkB toward repairing various adducts in different environments.