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Journal of Biochemistry Advance Access originally published online on September 18, 2007
Journal of Biochemistry 2007 142(5):647-654; doi:10.1093/jb/mvm177
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© 2007 The Japanese Biochemical Society

Cloning and Characterization of the yjeA Gene, Encoding a Novel Deoxyribonuclease, from Bacillus subtilis

Ka-Lun Ng1, Chui-Chi Lam1, Zhibiao Fu1, Yi-Fan Han1, Karl W.K. Tsim2 and Wan-Keung R. Wong1,*

1Department of Biochemistry; and 2Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

*To whom correspondence should be addressed at: Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. Tel: +852-2358-7299, Fax: +852-2358-1552, E-mail: bcwkrw{at}ust.hk

Received July 20, 2007; Accepted September 3, 2007


  Abstract

The yjeA gene, encoding a secreted protein, YjeA, of Bacillus subtilis, was cloned and characterized. A derivative of YjeA, the recombinant YjeA-H, which contained a C-terminal His6-tag, was purified from Escherichia coli for functional studies. YjeA-H was shown to be an endonuclease, which hydrolyses both single-stranded and double-stranded DNA, but not RNA. Covalently closed circular pBR322 DNA incubated with YjeA-H was shown by gel electrophoresis to be first nicked to an open circular form, and then to a linearized structure on a background of DNA smear, and finally to small species of linear molecules that accumulated gradually. When 32P-labelled pBR322 DNA was used as substrate, YjeA-H was shown to progressively nick both DNA strands in a random fashion, creating intermediates of various structures, as well as DNA smears comprising linear molecules of different sizes. The final products were found to consist essentially of degraded species of DNA. The detection of a putative signal peptide at the N-terminus of YjeA, together with the purification of YjeA-H from the culture supernatants of E. coli yjeA-H clones, and the identification of YjeA in the culture medium of Bacillus subtilis, supports the conclusion that YjeA is a secretory protein of Bacillus subtilis.

Key Words: DNA-specific, Escherichia coli, extracellular, gram positive bacterium, nicking endonuclease

Abbreviations: Ap, ampicillin; CCC, covalently closed circular; dsDNA, double-stranded DNA; OC, open circular; ORF, open reading frame; ssDNA, single-stranded DNA; Tc, tetracycline; YjeA-H, YjeA with a tag of six histidine residues at the C-terminus; [], denotes plasmid-carrier state


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