Probing 3'-ssDNA loop formation in E. coli RecBCD/RecBC-DNA complexes using non-natural DNA: a model for "Chi"...

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RecBC and RecBCD are essential helicases in Escherichia coli DNA repair.
• These enzymes process DNA double-strand breaks, utilizing 3'-single-stranded DNA tails.
• Previous hypotheses suggested 3'-ssDNA tails can loop out during RecBC/RecBCD binding.

Purpose of the Study:

• To investigate the role of 3'-ssDNA tail length in RecBC and RecBCD binding.
• To test the hypothesis of 3'-ssDNA tail looping upon helicase binding.
• To elucidate the mechanism of DNA end processing by RecBC/RecBCD.

Main Methods:

• Equilibrium binding assays using polyethylene glycol (PEG) spacers.
• Competition assays with Cy3-labeled DNA to quantify binding affinities.
• Studies with a RecBC mutant lacking the nuclease domain (RecB(Deltanuc)C).
• Computer modeling of RecBCD-DNA complexes.

Main Results:

• RecBC and RecBCD binding affinity is unaffected when PEG replaces ssDNA between the 6th and penultimate nucleotides.
• Replacing the terminal ssDNA nucleotides with PEG maximizes binding affinity, similar to a 3'-(dT)6 tail.
• 3'-ssDNA tail looping occurs even without the RecB nuclease domain, though it is stabilized by it.
• Computer modeling suggests 3'-ssDNA loop formation at the RecB/RecC interface.

Conclusions:

• The 3'-ssDNA tail can loop out in RecBC/RecBCD complexes, particularly when n is greater than or equal to 6 nucleotides.
• Loop formation is a key feature in the mechanism of RecBCD helicase action.
• A model is proposed for 3'-ssDNA loop formation at the RecB/RecC interface during Chi sequence recognition.