Repair of Methylation Damage in DNA and RNA by Mammalian AlkB Homologues

• Published in: The Journal of biological chemistry 2005-11, Vol.280 (47), p.39448-39459
• Main Authors: Lee, Dong-Hyun, Jin, Seung-Gi, Cai, Sheng, Chen, Yuan, Pfeifer, Gerd P., O'Connor, Timothy R.
• Format: Article
• Language: English
• Subjects: AlkB Homolog 1, Histone H2a Dioxygenase; Amino Acid Sequence; Animals; Base Sequence; DNA Damage; DNA Methylation; DNA Repair - genetics; DNA Repair - physiology; DNA Repair Enzymes; DNA, Complementary - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli; Genetic Complementation Test; Humans; In Vitro Techniques; Mice; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M509881200

Human and Escherichia coli derivatives of AlkB enzymes remove methyl groups from 1-methyladenine and 3-methylcytosine in nucleic acids via an oxidative mechanism that releases the methyl group as formaldehyde. In this report, we demonstrate that the mouse homologues of the α-ketoglutarate Fe(II) oxygen-dependent enzymes mAbh2 and Abh3 have activities comparable to those of their human counterparts. The mAbh2 and mAbh3 release modified bases from both DNA and RNA. Comparison of the activities of the homogenous ABH2 and ABH3 enzymes demonstrate that these activities are shared by both sets of enzymes. An assay for the detection of α-ketoglutarate Fe(II) dioxygenase activity using an oligodeoxyribonucleotide with a unique modification shows activity for all four enzymes studied and a loss of activity for eight mutant proteins. Steady-state kinetics for removal of methyl groups from DNA substrates indicates that the reactions of the proteins are close to the diffusion limit. Moreover, mAbh2 or mAbh3 activity increases survival in a strain defective in alkB. The mRNAs of AHB2 and ABH3 are expressed most in testis for ABH2 and ABH3, whereas expression of the homologous mouse genes is different. The mAbh3 is strongly expressed in testis, whereas highest expression of mAbh2 is in heart. Other purified human AlkB homologue proteins ABH4, ABH6, and ABH7 do not manifest activity. The demonstration of mAbh2 and mAbh3 activities and their distributions provide data on these mammalian homologues of AlkB that can be used in animal studies.