Interaction of human and bacterial AlkB proteins with DNA as probed through chemical cross-linking studies.
The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism. Three human homologs, ABH1, ABH2 and ABH3, have been identified, and two of them, ABH2 and ABH3, were shown to have similar repair activities to E.coli AlkB. However, ABH1 did not show any repair activity. It was suggested that ABH3 prefers single-stranded DNA and RNA substrates, whereas AlkB and ABH2 can repair damage in both single- and double-stranded DNA. We employed a chemical cross-linking approach to probe the structure and substrate preferences of AlkB and its three human homologs. The putative active site iron ligands in these proteins were mutated to cysteine residues. These mutant proteins were used to cross-link to different DNA probes bearing thiol-tethered bases. Disulfide-linked protein-DNA complexes can be trapped and analyzed by SDS-PAGE. Our results show that ABH2 and ABH3 have structural and functional similarities to E.coli AlkB. ABH3 shows preference for the single-stranded DNA probe. ABH1 failed to cross-link to the probes tested. This protein, unlike other AlkB proteins, does not seem to interact with DNA in its E.coli expressed form.
Mishina
Yukiko
Y
Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA.
Lee
Chih-Hui J
CH
He
Chuan
C
D000071498:AlkB Homolog 1, Histone H2a Dioxygenase; D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases; D003432:Cross-Linking Reagents; D015342:DNA Probes / Q000378:metabolism; D045643:DNA Repair Enzymes; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049308:Dioxygenases; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D015342:DNA Probes; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011506:Proteins; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C486439:ALKBH3 protein, human; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; C466427:AlkB protein, E coli; C499236:ALKBH1 protein, human; C486440:ALKBH2 protein, human; D000071498:AlkB Homolog 1, Histone H2a Dioxygenase; D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; C476001:ABHD1 protein, human; D002265:Carboxylic Ester Hydrolases; D045643:DNA Repair Enzymes