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Related Experiment Videos

DNA nucleotide sequence restricted by the RI endonuclease.

J Hedgpeth, H M Goodman, H W Boyer

    Proceedings of the National Academy of Sciences of the United States of America
    |November 1, 1972
    PubMed
    Summary

    Researchers determined the DNA sequence cleaved by the RI restriction endonuclease in coliphage lambda DNA. This analysis revealed a symmetrical four-nucleotide sequence (pAAT) at the cohesive termini generated by the enzyme.

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    Area of Science:

    • Molecular Biology
    • Enzymology
    • Genetics

    Background:

    • Restriction endonucleases are crucial tools in molecular biology for DNA manipulation.
    • Understanding the specific cleavage sites of these enzymes is essential for their application.
    • Coliphage lambda DNA serves as a model system for studying DNA-protein interactions.

    Purpose of the Study:

    • To determine the precise DNA base pair sequence recognized and cleaved by the RI restriction endonuclease.
    • To elucidate the structure of the cohesive termini generated by RI restriction enzyme activity.
    • To investigate the sequence symmetry associated with the RI endonuclease cleavage site.

    Main Methods:

    • Analysis of 5'-terminal (32)P-labeled nucleotides and oligonucleotides up to the heptamer from a pancreatic DNase digest of coliphage lambda DNA.

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  • Deduction of the nucleotide sequence adjacent to the RI break using data from the DNase digest.
  • Incorporation of 3'-dinucleotide sequence and nearest-neighbor analysis from repair synthesis using Rous sarcoma virus DNA polymerase.

    • Main Results:

    • The specific DNA sequence adjacent to the phosphodiester bonds cleaved by RI restriction endonuclease was identified.
    • RI endonuclease cleavage generates 5'-phosphoryls and short cohesive termini consisting of the four-nucleotide sequence (p)A(p)A(p)T(p)T.
    • The determined DNA sequence exhibits a striking palindromic symmetry.

    Conclusions:

    • The RI restriction endonuclease recognizes and cleaves a specific, symmetrical DNA sequence in coliphage lambda.
    • The enzyme produces defined four-nucleotide sticky ends, facilitating molecular cloning and genetic engineering applications.
    • The inherent symmetry of the recognition site is a key feature of RI endonuclease activity.