Neurospora endoexonuclease and its inactive (precursor?) form

Area of Science:

• Molecular Biology
• Enzymology
• Neurospora Genetics

Background:

• Two nuclease activities, single-strand specific endonuclease and strand nonspecific exonuclease, were previously observed to copurify from Neurospora mycleia.
• These activities were associated with a single polypeptide, suggesting a unified enzyme.

Purpose of the Study:

• To classify and characterize the enzyme responsible for the copurifying nuclease activities.
• To investigate the different forms and activation mechanisms of the endoexonuclease.

Main Methods:

• Purification of the inactive form of the endoexonuclease.
• Enzyme activity assays for endonuclease and exonuclease functions.
• Electrophoresis in 6 M urea-polyacrylamide gels to compare active and inactive forms.
• Octyl-Sepharose chromatography to assess protein properties.

Main Results:

• The enzyme was classified as an endoexonuclease, possessing both single-strand specific endonuclease and strand nonspecific exonuclease activities.
• Approximately 75% of the enzyme in log-phase extracts exists in an inactive form.
• The inactive form can be activated in vitro by proteases like trypsin or endogenous proteinases.
• Purification of the inactive form revealed it to be a distinct entity from the active enzyme, migrating slower on gels.
• The inactive form exhibits strong adsorption to octyl-Sepharose, indicating a hydrophobic domain.

Conclusions:

• The endoexonuclease is likely a single polypeptide with dual nuclease functions.
• The enzyme exists in an inactive precursor form (pronuclease) or as a complex with an inhibitor.
• Proteolytic activation is crucial for the enzyme's activity.
• The inactive form's properties suggest a potential precursor or inhibitor-bound state.