Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-loop Formation Than for Oligonucleotide Strand Exchange

Masayuki Takahashi
* ORCID logo ,
Axelle Renodon-Corniere
,
Tsutomu Mikawa
ORCID logo ,
Naoyuki Kuwabara
,
Kentaro Ito
ORCID logo ,
Dmitri Levitsky
,
Hiroshi Iwasaki
Version 1 : Received: 28 January 2024 / Approved: 29 January 2024 / Online: 30 January 2024 (03:41:28 CET)

A peer-reviewed article of this Preprint also exists.

Renodon-Corniere, A.; Mikawa, T.; Kuwabara, N.; Ito, K.; Levitsky, D.; Iwasaki, H.; Takahashi, M. Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange. Int. J. Mol. Sci. 2024, 25, 3633. Renodon-Corniere, A.; Mikawa, T.; Kuwabara, N.; Ito, K.; Levitsky, D.; Iwasaki, H.; Takahashi, M. Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange. Int. J. Mol. Sci. 2024, 25, 3633.

Abstract

Human RAD51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. The activator protein Swi5-Sfr1 and Ca2+ ions stimulate different reaction steps and induce a perpendicular orientation of DNA bases in the presynaptic complex. To investigate the importance 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) requires Ca2+ concentrations greater than 5 mM, while 1 mM is sufficient for strand exchange between two oligonucleotides. The structural change, which is evidenced by an increase in fluorescence intensity of poly(dεA) (a poly(dA) analog), reaches a plateau at 1 mM Ca2+. Meanwhile, the linear dichroism signal intensity at 260 nm, which is indicative of rigid perpendicular DNA base orientation, requires >2 mM Ca2+ for saturation and thus correlates 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 0.3 mM and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. We further discuss the parallels between Mg2+ activation of RecA and Ca2+ activation of HsRad51.

Keywords

Rad51 protein; homologous recombination; DNA strand exchange; Calcium ion; D-loop formation

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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