Genetic localization and characterization of a pKM101-coded endonuclease

Area of Science:

• Molecular Biology
• Enzymology
• Plasmid Biology

Background:

• The plasmid pKM101 is known to enhance mutagenesis.
• An associated endonuclease activity, resistant to EDTA, was observed.
• The function of this plasmid-encoded enzyme was previously unknown.

Purpose of the Study:

• To genetically and biochemically characterize the pKM101-mediated endonuclease.
• To determine the gene responsible for the nuclease activity and its location.
• To investigate the potential physiological role of this EDTA-resistant DNase.

Main Methods:

• Developed an assay using nondenaturing acrylamide gels with DNA to detect EDTA-resistant nuclease activity.
• Localized the gene for nuclease expression using plasmid DNA fragments.
• Determined gene transcription direction and protein molecular mass via cellular fractionation.

Main Results:

• Identified a 0.8-kilobase sequence on pKM101 responsible for nuclease expression, transcribed clockwise.
• The gene encodes a 16,000-dalton protein, identified as the nuclease itself.
• The enzyme is localized in the periplasm, but no direct physiological role in known plasmid phenotypes was found.

Conclusions:

• The pKM101 plasmid encodes its own EDTA-resistant endonuclease.
• This nuclease does not appear to directly mediate mutagenesis enhancement or other studied plasmid functions.
• Similar EDTA-resistant DNases were found in other plasmids, suggesting a broader phenomenon.