Published online 30 June 2004
Nucleic Acids Research, Vol. 32 No. 11 © Oxford University Press 2004; all rights reserved
Substrate specificities of bacterial and human AlkB proteins
1 Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, Rikshospitalet University Hospital, 0027 Oslo, Norway, 2 Department of Molecular Biosciences, University of Oslo, PO Box 1041, Blindern, 0316 Oslo, Norway and 3 Institute of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway
* To whom correspondence should be addressed at Department of Molecular Biosciences, University of Oslo, PO Box 1041, Blindern, 0316 Oslo, Norway. Tel: +47 22854840; Fax: +47 22854443; Email: pal.falnes{at}imbv.uio.no
Received April 30, 2004; Revised and Accepted May 28, 2004
Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.
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