Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1.
Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1.
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通过重组人脱嘌呤/脱嘧啶核酸内切酶 1 去除寡核苷酸底物中 DNA 链断裂损伤中的 3-磷酸乙醇酸。
DOI：
10.1093/nar/22.10.1866
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发表时间：
1994
影响因子：
14.9
通讯作者：
Jorgensen,TJ
中科院分区：
文献类型：
--
作者：
Winters,TA;Henner,WD;Russell,PS;McCullough,A;Jorgensen,TJ
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 oligodeoxy-ribonucleotide 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 Vmaxof 0.1 fmole/min/pg of HAP1 protein, and a Kmof 0.05 μM 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 byE.coliDNA 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 Vmaxand Kmfor the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 μM, respectively.
