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The AddAB helicase/nuclease forms a stable complex with its cognate chi sequence during translocation.

Frédéric Chédin1, Naofumi Handa, Mark S Dillingham

  • 1Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, California 95616, USA.

The Journal of Biological Chemistry
|April 25, 2006
PubMed
Summary

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The Bacillus subtilis AddAB enzyme transiently binds to its Chi sequence during DNA translocation. This interaction protects DNA from degradation, suggesting a regulatory mechanism for RecBCD-like enzymes.

Area of Science:

  • Molecular Biology
  • Enzymology
  • DNA Repair

Background:

  • The AddAB enzyme from Bacillus subtilis unwinds and degrades double-stranded DNA (dsDNA) via ATP-dependent helicase and nuclease activities.
  • It recognizes a specific DNA sequence, Chi, similar to the E. coli RecBCD enzyme, which attenuates its nuclease activity to generate single-stranded DNA (ssDNA) overhangs.
  • While RecBCD is known to pause at Chi, specific binding during translocation is not well-documented.

Purpose of the Study:

  • To investigate the interaction between the AddAB enzyme and its cognate Chi sequence (chi(Bs)) during DNA translocation.
  • To determine the nature and significance of AddAB binding to Chi.

Main Methods:

  • Demonstration of transient binding of AddAB to chi(Bs) during translocation using biochemical assays.

Related Experiment Videos

  • Assessment of the protective effect of AddAB binding on Chi-specific ssDNA against exonuclease I degradation.
  • Investigation of binding characteristics including non-covalent nature, affinity, and cis-preference through protein stripping and competitor assays.

    • Main Results:

    • AddAB enzyme exhibits transient binding to the chi(Bs) sequence during translocation.
    • This binding protects the 3'-end of chi(Bs)-specific ssDNA from degradation by exonuclease I.
    • The binding is non-covalent, occurs with high affinity only during translocation, and happens in cis.

    Conclusions:

    • The transient binding of AddAB to its Chi sequence is a key aspect of their interaction.
    • This molecular event likely underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes in DNA processing.