Nucleic Acids Research, 1994, Vol. 22, No. 10 1866-1873
© 1994
ENZYMOLOGY |
Removal of 3'-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1
Department of Radiation Medicine, Vincent T.Lombardi Cancer Research Center, Georgetown University Medical Center 3800 Reservoir Road NW, Washington, DC 20007 1Division of Hematology and Medical Oncology, Oregon Health Sciences University Portland, OR 97201, USA
*To whom correspondence should be addressed
Received December 30, 1993. Revised April 6, 1994. Accepted April 6, 1994.
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 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.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Takahashi, M. Tada, S. Igarashi, A. Koyama, H. Date, A. Yokoseki, A. Shiga, Y. Yoshida, S. Tsuji, M. Nishizawa, et al. Aprataxin, causative gene product for EAOH/AOA1, repairs DNA single-strand breaks with damaged 3'-phosphate and 3'-phosphoglycolate ends Nucleic Acids Res., June 28, 2007; 35(11): 3797 - 3809. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Sokhansanj and D. M. Wilson III Estimating the effect of human base excision repair protein variants on the repair of oxidative DNA base damage. Cancer Epidemiol. Biomarkers Prev., May 1, 2006; 15(5): 1000 - 1008. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Parsons, I. I. Dianova, and G. L. Dianov APE1 is the major 3'-phosphoglycolate activity in human cell extracts Nucleic Acids Res., July 6, 2004; 32(12): 3531 - 3536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-D. Junker, S. T. Hoehn, R. C. Bunt, V. Marathius, J. Chen, C. J. Turner, and J. Stubbe Synthesis, characterization and solution structure of tethered oligonucleotides containing an internal 3'-phosphoglycolate, 5'-phosphate gapped lesion Nucleic Acids Res., December 15, 2002; 30(24): 5497 - 5508. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. T. Hoehn, C. J. Turner, and J. Stubbe Solution structure of an oligonucleotide containing an abasic site: evidence for an unusual deoxyribose conformation Nucleic Acids Res., August 15, 2001; 29(16): 3413 - 3423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Unk, L. Haracska, S. Prakash, and L. Prakash 3'-Phosphodiesterase and 3'{right-arrow}5' Exonuclease Activities of Yeast Apn2 Protein and Requirement of These Activities for Repair of Oxidative DNA Damage Mol. Cell. Biol., March 1, 2001; 21(5): 1656 - 1661. [Abstract] [Full Text]
|
|
|
|
 |
|
 W. K. Hansen and M. R. Kelley Review of Mammalian DNA Repair and Translational Implications J. Pharmacol. Exp. Ther., October 1, 2000; 295(1): 1 - 9. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. J. DiBiase, Z.-C. Zeng, R. Chen, T. Hyslop, W. J. Curran Jr., and G. Iliakis DNA-dependent Protein Kinase Stimulates an Independently Active, Nonhomologous, End-Joining Apparatus Cancer Res., March 1, 2000; 60(5): 1245 - 1253. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. J. Prieto-Alamo and F. Laval Overexpression of the human HAP1 protein sensitizes cells to the lethal effect of bioreductive drugs Carcinogenesis, March 1, 1999; 20(3): 415 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Reardon, C. R. Lombardo, and M. Sander Drosophila Rrp1 Domain Structure as Defined by Limited Proteolysis and Biophysical Analyses J. Biol. Chem., December 18, 1998; 273(51): 33991 - 33999. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Masuda, R. A. O. Bennett, and B. Demple Rapid Dissociation of Human Apurinic Endonuclease (Ape1) from Incised DNA Induced by Magnesium J. Biol. Chem., November 13, 1998; 273(46): 30360 - 30365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-j. Xu, E. Y. Kim, and B. Demple Excision of C-4'-oxidized Deoxyribose Lesions from Double-stranded DNA by Human Apurinic/Apyrimidinic Endonuclease (Ape1 Protein) and DNA Polymerase beta J. Biol. Chem., October 30, 1998; 273(44): 28837 - 28844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Johnson, C. A. Torres-Ramos, T. Izumi, S. Mitra, S. Prakash, and L. Prakash Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites Genes & Dev., October 1, 1998; 12(19): 3137 - 3143. [Abstract] [Full Text]
|
|
|
|
|
|
D. M. W. III, M. Takeshita, A. P. Grollman, and B. Demple Incision Activity of Human Apurinic Endonuclease (Ape) at Abasic Site Analogs in DNA J. Biol. Chem., July 7, 1995; 270(27): 16002 - 16007. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Harrison, A. G. Ascione, D. M. Wilson III, and B. Demple Characterization of the Promoter Region of the Human Apurinic Endonuclease Gene (APE) J. Biol. Chem., March 10, 1995; 270(10): 5556 - 5564. [Abstract] [Full Text] [PDF]
|
|