Abstract
The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.
Highlights
RAD51 protein (Rad51) is a eukaryotic ortholog of RecA recombinase that is highly conserved among different species [1,2]
We have examined the effect of Swi5Sfr1 on the structure of the presynaptic filament, composed of fission yeast Rad51 (SpRad51), single-stranded DNA (ssDNA) and ATP, by flow linear dichroism (LD) spectroscopy
We have previously studied the structure of Rad51/DNA as well as RecA/DNA complex filaments [19,20,21], and recently we have demonstrated that the DNA bases in the human Rad51 (HsRad51)/ssDNA/ATP filament are oriented nearly perpendicular to the filament axis in the presence of Ca2+, almost no preferential orientation was observed in the presence of Mg2+ [22]
Summary
RAD51 protein (Rad51) is a eukaryotic ortholog of RecA recombinase that is highly conserved among different species [1,2]. It catalyzes the strand exchange reaction, which is an essential step in homologous recombination. The Swi5-Sfr heterodimer protein is one such accessory protein [10,11,12,13]. The protein is a heterodimer consisting of one Sfr protein and one Swi protein [12,15] bound noncovalently to each other by primarily two leucine zippers [16]. The shape of Swi5-Sfr, partially determined by X-ray crystallography [16], is long and somewhat crescent-shaped, making the protein fit well into the groove of the right Rad51-filament that surrounds
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