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Related Experiment Videos

Inhibitor evidence for allosteric interaction in the replitase multienzyme complex.

G P veer Reddy, A B Pardee

    Nature
    |July 7, 1983
    PubMed
    Summary

    Researchers found that thymidylate synthase is functionally linked to other DNA biosynthesis enzymes in hamster cells. Inhibitors targeting DNA synthesis enzymes affect thymidylate synthase activity within intact cells, suggesting complex interactions.

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    Area of Science:

    • Molecular Biology
    • Cell Biology
    • Biochemistry

    Background:

    • Previously identified a nuclear fraction ('replitase') from S phase Chinese hamster embryo fibroblasts (CHEF/18 cells) containing enzymes for DNA biosynthesis.
    • This replitase fraction comprises ~25 nm spherical particles, suggesting multienzyme complexes for de novo DNA synthesis.

    Purpose of the Study:

    • To investigate the functional association of thymidylate synthase with other enzymes within the DNA synthesizing complex.
    • To explore the impact of specific enzyme inhibitors on thymidylate synthase activity in intact cells versus soluble extracts.

    Main Methods:

    • Utilized specific inhibitors: hydroxyurea (ribonucleotide reductase), novobiocin (topoisomerase), and aphidicolin (DNA polymerase alpha).
    • Assessed the inhibition of thymidylate synthase activity in intact S phase CHEF/18 cells.

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  • Compared thymidylate synthase activity in soluble cell extracts under the same inhibitory conditions.

    • Main Results:

    • Hydroxyurea, novobiocin, and aphidicolin inhibited thymidylate synthase activity in intact CHEF/18 cells.
    • These inhibitors did not affect thymidylate synthase activity in soluble cell extracts.
    • Demonstrated functional association of thymidylate synthase with other DNA biosynthesis enzymes.

    Conclusions:

    • The results suggest allosteric interactions between subunits of the multienzyme DNA synthesizing complex.
    • These interactions are modulated by specific enzyme inhibitors in intact cells.
    • Supports the model of a coordinated, multienzyme complex for DNA replication.