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Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods
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Characterization of 3'----5' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus

V S Mikhailov, K A Marlyev, J O Ataeva

    Nucleic Acids Research
    |May 12, 1986
    PubMed

    Insights

    A silkworm virus enzyme possesses a unique 3' to 5' exonuclease activity specific for single-stranded DNA. This DNA polymerase-associated exonuclease releases deoxynucleoside monophosphates and functions distributively.

    Area of Science:

    • Molecular Biology
    • Virology
    • Enzymology

    Background:

    • Nuclear polyhedrosis viruses (NPVs) are significant insect pathogens.
    • Understanding viral DNA replication mechanisms is crucial for molecular biology.
    • The Bombyx mori NPV (BmNPV) is a well-studied baculovirus.

    Purpose of the Study:

    • To characterize the enzymatic activities of the DNA polymerase from Bombyx mori nuclear polyhedrosis virus (BmNPV Pol).
    • To investigate the presence and nature of associated exonuclease activity.
    • To determine the substrate specificity and kinetic properties of the identified exonuclease.

    Main Methods:

    • Purification of BmNPV Pol and associated enzymatic activities.
    • Ultracentrifugation in glycerol gradients to assess cosedimentation of activities.
    • Heat inactivation assays to compare polymerase and exonuclease stability.
    • Analysis of hydrolysis products to determine the mode of exonuclease action.

    Main Results:

    • BmNPV Pol copurified with a 3' to 5' exonuclease specific for single-stranded DNA.
    • No detectable 5' to 3' exonuclease activity was observed on single-stranded or duplex DNA.
    • The exonuclease activity released deoxynucleoside monophosphates from single-stranded DNA.
    • Polymerase and exonuclease activities exhibited identical heat inactivation kinetics and cosedimented during ultracentrifugation.
    • The exonuclease demonstrated strictly distributive hydrolysis of single-stranded DNA.

    Conclusions:

    • BmNPV Pol possesses an intrinsic 3' to 5' single-stranded DNA exonuclease activity.
    • This associated exonuclease activity is tightly linked to the DNA polymerase function.
    • The distributive nature of the exonuclease suggests a role in DNA repair or processing during viral replication.

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