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Action of ATP-dependent DNase from Hemophilus influenzae on cross-linked DNA molecules

M Orlosky, H O Smith

    The Journal of Biological Chemistry
    |October 10, 1976
    PubMed

    Insights

    The ATP-dependent DNase enzyme from Hemophilus influenzae exhibits resistance to cross-linked DNA. This enzyme binds to DNA cross-links, halting digestion but continuing ATP hydrolysis.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Enzymology

    Background:

    • The ATP-dependent DNase from Hemophilus influenzae is known to degrade double-stranded linear DNA.
    • This enzyme utilizes ATP hydrolysis to ADP for its exonucleolytic activity.

    Purpose of the Study:

    • To investigate the effect of DNA cross-links on the activity of the ATP-dependent DNase.
    • To understand the mechanism of DNA degradation and ATP hydrolysis in the presence of cross-linked DNA.

    Main Methods:

    • Enzymatic digestion assays using various cross-linked linear DNA molecules.
    • ATP hydrolysis rate measurements.
    • Sucrose gradient centrifugation to assess enzyme-DNA complex formation.

    Main Results:

    • Cross-linked DNA molecules, including Vaccinia DNA with natural terminal cross-links and phage T7 DNA with psoralen cross-links, showed partial resistance to digestion.
    • ATP hydrolysis continued even after DNA digestion ceased, indicating stable enzyme-DNA complex formation at cross-links.
    • A proposed mechanism involves initial exonucleolytic digestion to cross-links, followed by enzyme binding and sustained ATPase activity.

    Conclusions:

    • DNA cross-links act as barriers to the exonucleolytic activity of the ATP-dependent DNase.
    • The enzyme can form stable complexes at DNA cross-links, leading to persistent ATP hydrolysis.
    • This suggests a dual role for the enzyme involving both DNA processing and ATPase activity, potentially regulated by DNA structure.

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