# Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange

## TL;DR

Human Rad51 protein needs more calcium for D-loop formation than for strand exchange, revealing distinct roles of calcium in DNA repair.

## Contribution

The study reveals that calcium ions have two distinct roles in HsRad51-mediated DNA strand exchange reactions.

## Key findings

- D-loop formation requires Ca2+ concentrations above 5 mM, while oligonucleotide strand exchange needs only 1 mM.
- Structural changes in the presynaptic complex plateau at 1 mM Ca2+ but require higher concentrations for rigid DNA base orientation.
- HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, suggesting different steps are stimulated by different numbers of Ca2+ bonds.

## Abstract

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.