Cloning and characterization of the yjeA gene, encoding a novel deoxyribonuclease, from Bacillus subtilis.
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 His(6)-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 (32)P-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.
Ng
Ka-Lun
KL
Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Lam
Chui-Chi
CC
Fu
Zhibiao
Z
Han
Yi-Fan
YF
Tsim
Karl W K
KW
Wong
Wan-Keung R
WK
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial / Q000235:genetics*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D003851:Deoxyribonucleases; C527632:yjeA protein, E coli