Alpha-deoxyadenosine, a major anoxic radiolysis product of adenine in DNA, is a substrate for Escherichia coli endonuclease IV.
Oligonucleotides containing a unique alpha-deoxyadenosine or tetrahydrofuran (a model abasic site) were synthesized using phosphoramidite chemistry. Repair enzymes from Escherichia coli, including endonucleases III, IV, and VIII, exonuclease III, formamidopyrimidine N-glycosylase, and deoxyinosine 3'-endonuclease, as well as UV dimer N-glycosylases from T4 (den V) and Micrococcus luteus, were examined for their ability to recognize alpha-deoxyadenosine and tetrahydrofuran. In agreement with prior studies, a tetrahydrofuran-containing oligonucleotide was a substrate for endonuclease IV and exonuclease III, but not for the other repair enzymes. However, an oligonucleotide containing alpha-deoxyadenine was a substrate only for endonuclease IV. Competitive inhibition studies with both substrates confirmed that the activity recognizing alpha-deoxyadenine was endonuclease IV and not a possible contaminant in the endonuclease IV preparation. Using E. coli extracts, the activity that recognized alpha-deoxyadenine was dependent on nfo, the structural gene of endonuclease IV, further substantiating that endonuclease IV is the enzyme that recognized alpha-deoxyadenine. Kinetic measurements indicated that alpha-deoxyadenosine was as good a substrate for endonuclease IV as tetrahydrofuran; the Km and Vmax values for both substrates were similar. Using substrates that were labeled at either the 3'- or 5'-terminus, endonuclease IV was shown to hydrolyze the phosphodiester bond 5' to either alpha-deoxyadenosine or tetrahydrofuran, leaving the lesion, alpha-deoxyadenosine or tetrahydrofuran, on the 5'-terminus of the nicked site. The ability of endonuclease IV to recognize alpha-deoxyadenosine suggests that endonuclease IV is able to recognize a new class of DNA base lesions that is not recognized by other DNA N-glycosylases and AP endonucleases.
Ide
H
H
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Japan.
Tedzuka
K
K
Shimzu
H
H
Kimura
Y
Y
Purmal
A A
AA
Wallace
S S
SS
Kow
Y W
YW
D000225:Adenine / Q000737:chemistry; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003839:Deoxyadenosines / Q000737:chemistry* / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005663:Furans / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism
D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D005663:Furans; D009838:Oligodeoxyribonucleotides; C018674:tetrahydrofuran; D004247:DNA; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D000225:Adenine