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Related Experiment Videos

Cruciform-resolvase interactions in supercoiled DNA.

D M Lilley, B Kemper

    Cell
    |February 1, 1984
    PubMed
    Summary

    T4 endonuclease VII specifically cleaves short inverted repeats in supercoiled DNA, supporting the cruciform formation model. This enzyme acts as a probe for cruciform structures, which serve as Holliday junction models.

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    Area of Science:

    • Molecular Biology
    • Enzymology
    • Structural Biology

    Background:

    • T4 endonuclease VII is known to cleave Holliday-like junctions in DNA.
    • Short inverted repeats in supercoiled plasmids are cleaved by single-strand-specific nucleases.
    • Cruciform formation has been proposed to explain this site-specific cleavage.

    Purpose of the Study:

    • To investigate the specific cleavage of short inverted repeats by T4 endonuclease VII.
    • To provide further evidence for cruciform formation as the basis for this cleavage.
    • To explore the utility of T4 endonuclease VII as a probe for cruciform structures.

    Main Methods:

    • Cleavage assays using T4 endonuclease VII on supercoiled plasmids containing short inverted repeats.
    • Analysis of cleavage products to determine the precise locations of nicks.
    • Mapping of cleavage sites on specific cruciform sequences.

    Main Results:

    • T4 endonuclease VII specifically cleaves short inverted repeats, forming linear molecules with hairpin ends.
    • Cleavage occurs at positions corresponding to the stem-base of the cruciform structure.
    • Detailed mapping revealed nicks introduced 2-3 nucleotides from the sequence end on each strand.

    Conclusions:

    • The data strongly support the hypothesis that cruciform structures are formed by these inverted repeats.
    • T4 endonuclease VII can be used as a valuable tool to probe cruciform structures in DNA.
    • Short cruciform structures can serve as effective models for studying Holliday junctions.

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