Removal of 3′-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1

Thomas A. Winters, W. David Henner, Pamela S. Russell, Amanda McCullough, Timothy J. Jorgensen

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

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 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 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 μM, respectively.

Original languageEnglish (US)
Pages (from-to)1866-1873
Number of pages8
JournalNucleic Acids Research
Volume22
Issue number10
StatePublished - May 25 1994

Fingerprint

DNA-(Apurinic or Apyrimidinic Site) Lyase
DNA Breaks
Oligonucleotides
Endonucleases
DNA
Damage
Substrate
Oligodeoxyribonucleotides
Phosphate
Chemistry
Phosphates
Substrates
Proteins
Protein
Catalysis
Ionizing radiation
Nucleotides
Escherichia coli
Escherichia Coli
Hydrolysis

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

Removal of 3′-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1. / Winters, Thomas A.; Henner, W. David; Russell, Pamela S.; McCullough, Amanda; Jorgensen, Timothy J.

In: Nucleic Acids Research, Vol. 22, No. 10, 25.05.1994, p. 1866-1873.

Research output: Contribution to journalArticle

Winters, Thomas A. ; Henner, W. David ; Russell, Pamela S. ; McCullough, Amanda ; Jorgensen, Timothy J. / Removal of 3′-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1. In: Nucleic Acids Research. 1994 ; Vol. 22, No. 10. pp. 1866-1873.
@article{9247b1b959004334915523b9ce820869,
title = "Removal of 3′-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1",
abstract = "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 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 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 μM, respectively.",
author = "Winters, {Thomas A.} and Henner, {W. David} and Russell, {Pamela S.} and Amanda McCullough and Jorgensen, {Timothy J.}",
year = "1994",
month = "5",
day = "25",
language = "English (US)",
volume = "22",
pages = "1866--1873",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "10",

}

TY - JOUR

T1 - Removal of 3′-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1

AU - Winters, Thomas A.

AU - Henner, W. David

AU - Russell, Pamela S.

AU - McCullough, Amanda

AU - Jorgensen, Timothy J.

PY - 1994/5/25

Y1 - 1994/5/25

N2 - 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 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 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 μM, respectively.

AB - 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 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 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 μM, respectively.

UR - http://www.scopus.com/inward/record.url?scp=0028245076&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028245076&partnerID=8YFLogxK

M3 - Article

C2 - 7516064

AN - SCOPUS:0028245076

VL - 22

SP - 1866

EP - 1873

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 10

ER -