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Journal of Biochemistry Advance Access originally published online on April 13, 2009
Journal of Biochemistry 2009 146(2):201-208; doi:10.1093/jb/mvp060
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Reinvestigation of the Molecular Influence of Hypoxanthine on the DNA Cleavage Efficiency of Restriction Endonucleases BglII, EcoRI and BamHI

Akihiro Doi1,*, Seung Pil Pack2,*,{dagger}, Tsutomu Kodaki1 and Keisuke Makino1

1Institute of Advanced Energy, Kyoto University, Gokasho, Uji 611-0011, Japan; and 2Department of Biotechnology and Bioinformatics, Korea University, Jochiwon, Chungnam, 339-800, Korea

{dagger}To whom correspondence should be addressed. Tel: +82-041-860-1419, Fax: +82-041-864-2665, E-mail: spack{at}korea.ac.kr

Correspondence may also be addressed to Keisuke Makino. Tel: +81-0774-38-3517, Fax: +81-0774-38-3524, E-mail: kmak{at}iae.kyoto-u.ac.jp

Received December 22, 2008; Accepted March 25, 2009


  Abstract

Hypoxanthine (Hyp), a deaminated base of adenine (Ade), can be employed as a good probe molecule to reveal the significance of the minor groove of guanine (Gua) in biomolecular interactions because Hyp possesses a similar structure to Gua lacking its 2-amino group. In this study, we examined cleavage efficiencies of restriction endonuclease enzymes on DNA substrates with Hyp in their recognition sequences. As a substrate for BglII, EcoRI and BamHI, 24-mer DNA oligomer with Hyp (in place of Gua) was prepared together with its complementary sequences with cytosine (Cyt) or thymine (Thy) as the counter base. At 37°C incubation for 1 h, BglII and EcoRI showed higher DNA cleavage reactivity on Hyp-containing DNA substrates than on normal ones, whereas BamHI showed lower values on Hyp-containing substrates. Such high cleavage performance of BglII and EcoRI on Hyp-containing DNA substrates is in contrast to the results obtained 20 years ago, in which short DNA substrates (8- or 10-mer) and low reaction temperatures (15–20°C) were employed. These new results suggest that the lack of the exocyclic 2-amino group of Gua could contribute to enhanced recognition access of BglII and EcoRI to DNA substrates.

Key Words: BglII, EcoRI, hypoxanthine, minor groove, restriction endonuclease

Abbreviations: Hyp (H), hypoxanthine; Gua (G), guanine; Ade (A), adenine; Cyt (C), cytosine; Thy (T), thymine; Ino (I), inosine; Tm, melting temperature

*These authors contributed equally to this work.


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