title: Removal of N-alkyl modifications from N(2)-alkylguanine and N(4)-alkylcytosine in DNA by the adaptive response protein AlkB.
abstract: The AlkB enzyme is an Fe(II)- and α-ketoglutarate-dependent dioxygenase that repairs DNA alkyl lesions by a direct reversal of damage mechanism as part of the adaptive response in E. coli. The reported substrate scope of AlkB includes simple DNA alkyl adducts, such as 1-methyladenine, 3-methylcytosine, 3-ethylcytosine, 1-methylguanine, 3-methylthymine, and N(6)-methyladenine, as well as more complex DNA adducts, such as 1,N(6)-ethenoadenine, 3,N(4)-ethenocytosine, and 1,N(6)-ethanoadenine. Previous studies have revealed, in a piecemeal way, that AlkB has an impressive repertoire of substrates. The present study makes two additions to this list, showing that alkyl adducts on the N(2) position of guanine and N(4) position of cytosine are also substrates for AlkB. Using high resolution ESI-TOF mass spectrometry, we show that AlkB has the biochemical capability to repair in vitro N(2)-methylguanine, N(2)-ethylguanine, N(2)-furan-2-yl-methylguanine, N(2)-tetrahydrofuran-2-yl-methylguanine, and N(4)-methylcytosine in ssDNA but not in dsDNA. When viewed together with previous work, the experimental data herein demonstrate that AlkB is able to repair all simple N-alkyl adducts occurring at the Watson-Crick base pairing interface of the four DNA bases, confirming AlkB as a versatile gatekeeper of genomic integrity under alkylation stress.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Deyu
authors.initials: D
authors.lastname: Li
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Fedeles
authors.forename: Nidhi
authors.initials: N
authors.lastname: Shrivastav
authors.forename: James C
authors.initials: JC
authors.lastname: Delaney
authors.forename: Xuedong
authors.initials: X
authors.lastname: Yang
authors.forename: Cintyu
authors.initials: C
authors.lastname: Wong
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Drennan
authors.forename: John M
authors.initials: JM
authors.lastname: Essigmann
mesh_terms: D000478:Alkylation; D020029:Base Pairing; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009841:Oligonucleotides / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D018736:DNA Adducts; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D006147:Guanine; D003596:Cytosine; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli