UVM Theses and Dissertations
Format:
Print
Author:
Finstad, Kristiaan H.
Title:
Dept./Program:
Biochemistry
Year:
2010
Degree:
MS
Abstract:
Activities of the RecA recombinase enable the radiation-resistant bacterium Deinococcus radiodurans to survive catastrophic DNA damage induced by extreme levels of ionizing radiation. D.radiodurans RecA exhibits high sequence and structural conservation with other members of the RecA family, but its recombination mechanism appears to be unique: evidence suggests that unlike other recombinases, D. radiodurans RecA prefers to initiate DNA strand exchange reactions by assembling into filaments on double-stranded rather than single-stranded DNA.
To better understand the roles of protein-dsDNA and protein-ssDNA interactions in the strand exchange mechanism of D.radiodurans RecA, we mutated a conserved aromatic residue, Tyr-215, in its major DNA binding loop, L2, and characterized the biochemical properties of these mutants in comparison with wild-type RecA. The non-conservative mutant RecA-Y215A exhibits reduced affinity for both ssDNA and dsDNA, and is a weak ATPase and strand exchange enzyme. In contrast, the conservative mutant RecA-Y215W exhibits DNA binding activities similar to wild-type, and is a strong ATPase and strand exchange enzyme.
Salt effects on the DNA strand exchange activities of wild-type and mutant enzymes correlate well with salt effects on protein-dsDNA interactions, consistent with a common protein-dsDNA intermediate preceding the rate-limiting step. Differential responses of wild-type and mutant enzyme activities to single-strand DNA-binding protein (SSB) argue for a post-synaptic role of SSB in strand exchange. Effects of DNA and nucleotides on RecA-Y215W tryptophan fluorescence suggest that L2 serves as part of a flexible primary DNA binding site that can accommodate dsDNA as well as ssDNA.
To better understand the roles of protein-dsDNA and protein-ssDNA interactions in the strand exchange mechanism of D.radiodurans RecA, we mutated a conserved aromatic residue, Tyr-215, in its major DNA binding loop, L2, and characterized the biochemical properties of these mutants in comparison with wild-type RecA. The non-conservative mutant RecA-Y215A exhibits reduced affinity for both ssDNA and dsDNA, and is a weak ATPase and strand exchange enzyme. In contrast, the conservative mutant RecA-Y215W exhibits DNA binding activities similar to wild-type, and is a strong ATPase and strand exchange enzyme.
Salt effects on the DNA strand exchange activities of wild-type and mutant enzymes correlate well with salt effects on protein-dsDNA interactions, consistent with a common protein-dsDNA intermediate preceding the rate-limiting step. Differential responses of wild-type and mutant enzyme activities to single-strand DNA-binding protein (SSB) argue for a post-synaptic role of SSB in strand exchange. Effects of DNA and nucleotides on RecA-Y215W tryptophan fluorescence suggest that L2 serves as part of a flexible primary DNA binding site that can accommodate dsDNA as well as ssDNA.