HsRAD51(F129V) and HsRAD51(H294V) are deficient in D-loop formation and strand exchange.

Abstract

<p>(<b>A</b>) <i>In vitro</i> D-loop assay reaction schematic. (<b>B</b>) 0.8 µM of HsRAD51, HsRAD51(F129V), or HsRAD51(H294V) and [P³²]-labeled ssDNA (90mer; 2.4 µM nt) were preincubated for 10 min at 37°C in the reaction buffer containing 1 mM ATP and 1 mM MgCl₂ or CaCl₂. Reactions were initiated by the addition of supercoiled pBS SK(-) plasmid DNA (35 µM bp). After 15 min, reactions were terminated by the addition of proteinase-K and SDS. Joint molecules (JMs) were analyzed on a 0.9% agarose gel. (<b>C</b>) Analysis of salt and RPA requirement for strand exchange. Reaction schematic shown above: HsRAD51 (5 µM) and φX174 circular ssDNA (30 µM nt) were pre-incubated with 2.5 mM ATP and 1 mM MgCl₂ at 37°C for 5 m prior to the addition of 150 mM NaNH₄HPO₄ (if indicated) and linear φX174 dsDNA (15 µM bp). After 5 m, HsRPA (2 µM) was added (if indicated) and the incubation was continued for 3 h. Samples were deproteinized and analyzed on 0.9% agarose gel with 0.1 µg/mL ethidium bromide.</p