Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1.
A recombinant human AP endonuclease, HAP1, was constructed and characterized with respect to its ability to recognize and act upon a model double-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3'-phosphoglycolate and 5'-phosphate end-group chemistries. This oligodeoxyribonucleotide substrate exactly duplicates the chemistry and configuration of a major DNA lesion produced by ionizing radiation. HAP1 was found to recognize the strand break, and catalyze the release of the 3'-phosphoglycolate as free phosphoglycolic acid. The enzyme had a Vmax of 0.1 fmole/min/pg of HAP1 protein, and a Km of 0.05 microM for the 3'-phosphoglycolate strand break lesion. The mechanism of catalysis was hydrolysis of the phosphate ester bond between the 3'-phosphoglycolate moiety and the 3'-carbon of the adjacent dGMP moiety within the oligonucleotide. The resulting DNA contained a 3'-hydroxyl which supported nucleotide incorporation by E. coli DNA polymerase I large fragment. AP endonucleolytic activity of HAP1 was examined using an analogous double-stranded 39-mer oligodeoxyribonucleotide substrate, in which the strand break site was replaced by an apyrimidinic site. The Vmax and Km for the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 microM, respectively.
Winters
T A
TA
Department of Radiation Medicine, Vincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, Washington, DC 20007.
Henner
W D
WD
Russell
P S
PS
McCullough
A
A
Jorgensen
T J
TJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006016:Glycolates / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D003854:Deoxyribonucleotides; D006016:Glycolates; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C013193:phosphoglycolate