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Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry
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The single-stranded DNA-binding protein of Deinococcus radiodurans
Julie Malia Eggington1, Nami Haruta, Elizabeth Anne Wood
1Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA. jeggington@biochem.wisc.edu
BMC Microbiology
|January 14, 2004
Summary
The Deinococcus radiodurans ssb gene was corrected, revealing a contiguous open reading frame for the largest bacterial SSB monomer. This protein, functioning as a homodimer, efficiently stimulates DNA strand exchange, aiding understanding of radiation resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Deinococcus radiodurans R1 exhibits remarkable radiation resistance and DNA repair capabilities.
- Single-stranded DNA-binding (SSB) protein is crucial for DNA replication, recombination, and repair in all organisms.
- Previous genomic data suggested a complex, frameshifting mechanism for D. radiodurans SSB synthesis.
Purpose of the Study:
- To re-sequence and correct the Deinococcus radiodurans ssb gene.
- To characterize the D. radiodurans SSB protein and its function.
- To elucidate the role of SSB in the DNA repair mechanisms of D. radiodurans.
Main Methods:
- Re-sequencing of the Deinococcus radiodurans ssb gene region.
- Bioinformatic analysis of the corrected open reading frame (ORF).
- Protein purification and biochemical assays, including RecA-promoted DNA strand exchange.
Main Results:
- Correction of sequencing errors revealed a single, contiguous ssb ORF, eliminating the need for frameshifting.
- The corrected gene encodes the largest bacterial SSB monomer to date, featuring two oligonucleotide/oligosaccharide-binding (OB) folds.
- The purified SSB protein functions as a homodimer and efficiently stimulates RecA-mediated DNA strand exchange.
Conclusions:
- The Deinococcus radiodurans ssb gene is a contiguous ORF encoding a large SSB monomer.
- The SSB protein functions as a homodimer and effectively promotes DNA strand exchange.
- This finding provides a key component for understanding the exceptional DNA repair capacity of Deinococcus radiodurans.
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