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Related Experiment Videos

Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates.

P Bedinger1, M Munn, B M Alberts

  • 1Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.

The Journal of Biological Chemistry
|October 5, 1989
PubMed
Summary
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Bacteriophage T4 DNA polymerase pausing during synthesis is influenced by DNA structure and accessory proteins. T4 gene 41 helicase eliminates pausing, unlike T4 dda helicase, suggesting specific protein interactions regulate DNA replication fork movement.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • DNA synthesis by bacteriophage T4 DNA polymerase holoenzyme can exhibit sequence-specific pausing.
  • The T4 gene 32 protein (helix destabilizing protein) is involved in this pausing phenomenon.
  • Pausing sites can occur in regions with predicted secondary structures (hairpin helices) or those without obvious secondary structures.

Purpose of the Study:

  • To investigate the mechanisms underlying sequence-specific pausing during bacteriophage T4 DNA polymerase-catalyzed DNA synthesis.
  • To determine the effect of accessory proteins, specifically T4 helicases (gene 41 and dda proteins), on replication fork pausing.

Main Methods:

  • Characterization of DNA synthesis pausing by T4 DNA polymerase holoenzyme and T4 gene 32 protein on a bacteriophage fd DNA template.

Related Experiment Videos

  • Determination of the precise locations of DNA chain ends at pause sites using nucleotide resolution.
  • Analysis of the impact of adding T4 gene 41 protein (DNA helicase) or T4 dda protein (DNA helicase) to the replication system.

    • Main Results:

    • Sequence-specific pausing was observed, with some sites correlating with predicted hairpin structures and others not.
    • A clustering of pause sites was detected, suggesting polymerase holoenzyme destabilization in specific DNA regions.
    • Addition of T4 gene 41 protein significantly increased replication fork rate and eliminated pausing, while T4 dda protein did not affect pausing.

    Conclusions:

    • Replication fork pausing by T4 DNA polymerase is influenced by DNA sequence and structure, as well as accessory proteins.
    • The T4 gene 41 helicase effectively resolves pausing, potentially through specific interactions with the polymerase or processive movement along DNA.
    • The differential effects of T4 gene 41 and dda proteins highlight distinct roles of helicases in regulating DNA replication fidelity and processivity.