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Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD-DNA complexes.

Ashley R Carter1, Maasa H Seaberg2, Hsiu-Fang Fan3

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The RecBCD enzyme exhibits dynamic DNA binding during unwinding, not uniform steps. This motion, influenced by ATP and DNA structure, reveals conformational changes essential for its helicase function.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Enzymology

Background:

  • RecBCD is a crucial enzyme with helicase and nuclease functions.
  • Understanding its helicase mechanism is vital for DNA repair and replication.
  • Previous studies lacked high-resolution insights into RecBCD's unwinding dynamics.

Purpose of the Study:

  • To investigate the precise mechanism of RecBCD helicase activity at low ATP concentrations.
  • To characterize the nature of DNA unwinding steps and associated enzyme dynamics.
  • To elucidate the role of ATP binding and DNA structure in RecBCD function.

Main Methods:

  • Utilized a high-precision optical-trapping assay (1 bp resolution) to measure RecBCD unwinding.
  • Analyzed DNA length fluctuations and enzyme motion under controlled ATP conditions (2-4 μM).
  • Investigated the effects of DNA sequence (GC-content), strand gaps, and ATP analogs on RecBCD dynamics.

Main Results:

  • Observed non-uniform DNA unwinding, characterized by rapid, large-scale (∼4 bp) length variations, not uniform steps.
  • These dynamics were present even without ATP and were linked to an unwinding-competent state requiring a 5' strand.
  • Dynamics were modulated by DNA GC-content and suppressed by DNA cross-links or non-hydrolyzable ATP analogs.

Conclusions:

  • RecBCD engages DNA in a dynamic, conformational mode during unwinding, involving enzyme-DNA complex fluctuations.
  • This dynamic binding is intrinsically linked to the enzyme's helicase function and is modulated by the nucleotide state of its ATP-binding pocket.
  • The findings provide a new mechanistic perspective on RecBCD's role in DNA processing.