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Related Experiment Videos

Discontinuous DNA replication in mouse P-815 cells.

J R Gautschi, J M Clarkson

    European Journal of Biochemistry
    |January 2, 1975
    PubMed
    Summary

    DNA strand elongation occurs discontinuously via Okazaki fragments at both DNA ends. These fragments are primarily double-stranded, not single-stranded, and lack ribonucleotides at their 5

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    Biochemistry·1998

    Area of Science:

    • Molecular Biology
    • Genetics
    • Biochemistry

    Background:

    • Understanding DNA replication mechanisms is crucial for cell biology.
    • The nature and structure of newly synthesized DNA strands, including Okazaki fragments, are key areas of investigation.
    • Previous research suggested Okazaki fragments as intermediates in DNA synthesis.

    Purpose of the Study:

    • To analyze the length and structure of newly synthesized DNA strands in mouse P-815 cells.
    • To investigate the discontinuous nature of DNA strand elongation and the characteristics of Okazaki fragments.
    • To determine if ribonucleotides are present at the 5' end of Okazaki fragments.

    Main Methods:

    • Analysis of newly synthesized DNA strand length using electrophoresis and alkaline sucrose gradient sedimentation.
    • Pulse-labeling with [3-H]-thymidine under varying temperature and time conditions.
    • Assessment of Okazaki fragment structure using single-strand-specific nuclease S-1 and isopycnic gradient centrifugation.
    • Investigation of 5' end composition using isopycnic CsCl/Cs2SO4 gradients and polynucleotide kinase assay.

    Main Results:

    • Short DNA strands (Okazaki fragments) of 50-200 nucleotides were predominantly labeled with [3-H]thymidine.
    • Approximately 90% of Okazaki fragments were resistant to single-strand-specific nuclease S-1, indicating double-stranded character.
    • No evidence of ribonucleotides at the 5' end of Okazaki fragments was found.

    Conclusions:

    • DNA strand elongation occurs discontinuously through Okazaki fragments at both 5' and 3' ends.
    • Okazaki fragments are predominantly double-stranded molecules, not single-stranded as previously hypothesized.
    • The 5' end of Okazaki fragments does not appear to contain ribonucleotides.

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