A continuous kinetic assay for RecA-mediated DNA strand exchange using DNA-based flourescent signal
Singleton, Scott F.
Master of Arts
A continuous kinetic assay for RecA-mediated DNA strand exchange using a DNA-based fluorescent signal has been successfully developed and employed. 2-Aminopurine deoxyribonucleoside phosphoradmite was synthesized and incorporated into oligonuceleotides. The ODNs were HPLC-purified and gave excellent time-dependent spectrophtometric data on DNA hybridization. Active RecA protein was overexpressed and purified. The activation of the RecA protein for ATP hydrolysis by single-stranded DNA was investigated using this assay. From our preliminary kinetic data, the assay provides a view of the discrete steps along the reaction pathway. The first observation of a direct isomerization pathway for RecA monomers associated with DNA was accomplished. A mechanistic model in which association of RecA-ATP to single-stranded DNA is turnover-limiting was established. Synthesis of fluorescent nucleoside triphosphate is underway and expected to give satisfactory results.
Biochemistry; Organic chemistry