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Nucleic Acids Research, 1993, Vol. 21, No. 21 4867-4872
© 1993

ENZYMOLOGY

Genetic and biochemical analysis of an endonuclease encoded by the IncN plasmid pKM101

Robert F. Pohlman+, Franklin Liu§, Lu Wang, Margret I. Moré and Stephen C. Winans*

Section of Microbiology, Cornell University Ithaca, NY 14853, USA

*To whom correspondence should be addressed

Received August 16, 1993. Revised September 13, 1993. Accepted September 13, 1993.

The IncN plasmid pKM101 nuc gene encodes a periplasmically localized endonuclease. DNA sequence analysis indicates that this gene encodes a hydrophilic protein of about 19.5 kDa containing a hydrophobic signal sequence, nuc is homologous to a partially sequenced open reading frame adjacent to the sog gene of the plasmid CollB-P9, a plasmid known to encode an endonuclease similar to that of pKM101. A partially sequenced tra gene directly upstream of nuc is homologous to the virB11 gene of Agrobacterium tumefaciens. We have partially purified the pKM101 nuclease by osmotic shock and cation exchange chromatography, and used this enzyme preparation to sequence the protein's amino terminus. The first 13 amino acids of the mature protein match amino acids 23 to 35 of the predicted sequence, indicating that the protein is proteolytically processed to a molecular mass of approximately 17 kDa, probably during export to the periplasmic space. The enzyme was able to attack many sites along an end labelled duplex DNA substrate, but showed clearly preferred cleavage sites, and may cleave preferentially at purine-rich regions.

+Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

§Present address: Cornell University College of Medicine, New York, NY 10021, USA


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