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Base flipping in nucleotide excision repair.

Erik Malta¹, Geri F Moolenaar, Nora Goosen

¹Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands.

The Journal of Biological Chemistry

|November 12, 2005

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Bacterial DNA repair protein UvrB binds DNA lesions by flipping bases into a protein pocket. Cofactor binding (ADP/ATP) influences DNA conformational changes and complex stability, crucial for repair incision.

Area of Science:

Molecular Biology
Biochemistry
DNA Repair Mechanisms

Background:

UvrB is a key protein in bacterial nucleotide excision repair, recognizing diverse DNA lesions.
A beta-hairpin structure within UvrB is critical for its damage-specific DNA binding.
Understanding UvrB's interaction with DNA at a molecular level is essential for elucidating repair pathways.

Purpose of the Study:

To investigate DNA conformational changes induced by UvrB binding to damaged DNA.
To determine the role of UvrB's beta-hairpin in DNA lesion recognition and base flipping.
To explore how cofactor binding (ADP/ATP) affects UvrB-DNA complex stability and DNA conformation.

Main Methods:

Utilized the fluorescent adenine analogue 2-aminopurine to monitor DNA conformational alterations.

Related Experiment Videos

Studied UvrB-DNA complexes with cholesterol-damaged DNA fragments.

Analyzed base accessibility using acrylamide quenching and monitored fluorescence changes.

Main Results:

UvrB binding to damaged DNA causes the adjacent 3' base to flip into an extrahelical position within a protein pocket.
The base opposite the flipped base is also extruded from the DNA helix.
Cofactor binding significantly influences the degree of base solvent exposure and UvrB-DNA complex stability.
The hairpin residue Tyr-95 is implicated in flipping the base on the non-damaged strand.

Conclusions:

UvrB induces significant DNA conformational changes, including base flipping, upon binding to lesions.
Cofactor-dependent modulation of UvrB-DNA interactions impacts repair efficiency.
The observed conformational changes, particularly base flipping in the non-damaged strand, are likely important for subsequent incision by UvrC.