Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3' endonuclease, from Escherichia coli.
We have purified a novel endonuclease from Escherichia coli that recognizes deoxyinosine, a deamination product of deoxyadenosine in DNA. This activity, which we named deoxyinosine 3' endonuclease, is different from the known hypoxanthine DNA N-glycosylases which have been partially characterized in E. coli and other organisms. The enzyme was purified 24,800-fold to apparent homogeneity. SDS- and activity PAGE analyses indicate that the enzyme has an apparent molecular mass of 25 kDa. Deoxyinosine 3' endonuclease recognized deoxyinosine in both single- and double-stranded DNA but exhibited a 4-fold preference for double stranded over single-stranded DNA. In addition to deoxyinosine, the enzyme recognized urea residues and AP sites. Deoxyinosine 3' endonuclease has an obligatory requirement for Mg2+, but other cations such as Co2+ and Mn2+ could partially replace Mg2+. The optimal pH for deoxyinosine 3' endonuclease was around 7.5. In contrast to most of the known repair enzymes, deoxyinosine 3' endonuclease makes an incision at the second phosphodiester bond 3' to a deoxyinosine or AP site, leaving behind the intact lesion on the nicked DNA. Therefore, deoxyinosine 3' endonuclease recognizes, but does not remove, the lesion from the DNA molecule. The biological significance of this novel activity is discussed with reference to other repair activities in E. coli.
Yao
M
M
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405.
Hatahet
Z
Z
Melamede
R J
RJ
Kow
Y W
YW
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007288:Inosine / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D012330:RNA, Double-Stranded; D007288:Inosine; D004706:Endodeoxyribonucleases; C012271:deoxyinosine