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© 1989 Oxford University Press

research-article

Repair of O6-methylguanine, O6-ethylguanine, O6-isopropylguanine, and O4-methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase

Robert J. Graves 1, Benjamin F.L. Li and Peter F. Swann

Department of Biochemistry, University College and Middlesex School of Medicine London WCIE 6BT, UK
1Present address: Department of Physiology, Milton S.Hershey Medical Center Hershey, PA 17033, USA

Self-complementary oligodeoxynucleotides have been synthesized containing O6-methylguanine (O6meG), O6-ethylguanine (O6etG), O6-isopropylguanine (O6iprG) and O4-methylthymine (O4meT). They anneal in solution to give double-stranded DNA. These double helices have been used as substrates for the DNA repair protein O6-alkyl-guanine-DNA-alkyltransferase coded for by the ada gene of Escherichia coli. The repair followed second-order chemical kinetics. O6meG was repaired by the 19-kd transferase at a rate of 2.54x107 M–1 s–1 which is close to the theoretical limit for a diffusion-controlled reaction; O6etG and O4meT are repaired 1000 and 10000 times more slowly. The 39-kd alkyltransferase (which is precursor to the 19-kd form) and the 19-kd transferase repaired O6etG at similar rates. O6iprG was not repaired. The repair of oligomers containing O6meG was only slightly inhibited by the presence of non-alkylated oligomers. Oligomers containing O6etG were only slightly more effective as inhibitors of repair than the non-alkylated oligomers, indicating that the transferase does not bind selectively to alkylated DNA. Parallel structural studies have shown that O6-alkylguanine:C and O4-alkylthymine:A base pairs have a similar geometry with the alkylated base displaced into the major groove of the DNA in contrast to O6-alkylguanine:T and O4-alkylthymine:G base pairs which retain the Watson-Crick alignment with N1 of the purine juxtaposed to N3 of the pyrimidine. Measurement of the rate of repair of these different base pairs suggests that pairs with the alkyl group exposed in the major groove may be repaired more rapidly than those with the alkyl group more deeply buried in the helix.


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Home page
 Nucleic Acids ResHome page
P. E. Verdemato, J. A. Brannigan, C. Damblon, F. Zuccotto, P. C. E. Moody, and L.-Y. Lian
DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase
Nucleic Acids Res., October 1, 2000; 28(19): 3710 - 3718.
[Abstract] [Full Text] [PDF]


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