Nucleic Acids Research Advance Access originally published online on September 13, 2006
Nucleic Acids Research 2006 34(17):4743-4751; doi:10.1093/nar/gkl553
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Nucleic Acids Research, 2006, Vol. 34, No. 17 4743-4751
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis
Department of Applied Biological Science, Nihon University College of Bioresource Sciences Fujisawa-shi, Kanagawa 252-8510, Japan
*To whom correspondence should be addressed. Tel: +81 466 84 3350; Fax: +81 466 84 3698; Email: htakaha{at}brs.nihon-u.ac.jp
Received June 12, 2006. Revised July 10, 2006. Accepted July 18, 2006.
Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5' phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5'-dTdCdA-3' but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5'-dTdCdA-3', which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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