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

Abstract

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 AlkB and its human homologs, 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 on repairing various adducts in different environments.

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

“…The three proteins were then expressed in E. coli hosts, isolated and purified by affinity chromatography as described in Experimental Section. 55 …”

“…59 , 60 The k cat /K M values of AlkB repair confirm that the enzyme prefers to repair m1A and m3C in ss-DNA as compared with ds-DNA. 55 …”

“…24,30,55 We tested the repair activity in both ss-DNA and ds-DNA substrates in this study. The experimental results reported in this paper provide a strong kinetic basis for the previous observations.…”

“…43,55 The complementary 23mer oligonucleotides were synthesized with the sequence of 5′-CTGGGACGCCYAGGTCTTCACTG-3′, where Y represents the position incorporating the regular bases T or G, and named as 23mer-Tcp or 23mer-Gcp. The 23mer oligonucleotides complementary to 23mer-Tcp and 23mer-Gcp were also synthesized, with the sequence 5′-CAGTGAAGACCTZGGCGTCCCAG-3′, where Z was the regular base A or C, named as 23mer-A or 23mer-C. All DNA syntheses employed solid-phase phosphoramidite chemistry performed on a MerMade-4 Oligonucleotide Synthesizer.…”

“…The 23mer oligonucleotides complementary to 23mer-Tcp and 23mer-Gcp were also synthesized, with the sequence 5′-CAGTGAAGACCTZGGCGTCCCAG-3′, where Z was the regular base A or C, named as 23mer-A or 23mer-C. All DNA syntheses employed solid-phase phosphoramidite chemistry performed on a MerMade-4 Oligonucleotide Synthesizer. 55 The oligonucleotides were purified by HPLC (Thermo Fisher Scientific) on a DNAPac PA-100 Semi-Preparative column (Phenomenex). Solvent A was 100 mM 1:1 triethylamine-acetic acid (TEAA) in water and solvent B was 100% acetonitrile.…”