Elsevier

Biochimie

Volume 92, Issue 12, December 2010, Pages 1832-1838
Biochimie

Research paper
Targeting human Rad51 by specific DNA aptamers induces inhibition of homologous recombination

https://doi.org/10.1016/j.biochi.2010.08.006Get rights and content

Abstract

Human Rad51 (HsRad51), a key element of the homologous recombination repair pathway, is related to the resistance of cancer cells to chemo- and radio-therapies. This protein is thus a good target for the development of anti-cancer treatments. We have searched for new inhibitors directed against HsRad51 using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach. We have selected three aptamers displaying strong effects on strand exchange activity. Analysis by circular dichroism shows that they are highly structured DNA molecules. Our results also show that they affect the first step of the strand exchange reaction by promoting the dissociation of DNA from the ATP/HsRad51/DNA complex. Moreover, these inhibitors bind only weakly to RecA, a prokaryotic ortholog of HsRad51. Both the specificity and the efficiency of their inhibition of recombinase activity offer an analytical tool based on molecular recognition and the prospect of developing new therapeutic agents.

Introduction

Homologous recombination (HR) is an evolutionarily conserved mechanism for the repair of double-strand breaks in DNA, and is also involved in DNA segregation and the creation of gene diversity [1], [2], [3], [4], [5], [6]. A key component of HR in eukaryotes is the protein Rad51, which catalyzes strand exchange between homologous DNA.

This protein acts against radio- and chemo-therapies by repairing the DNA damage caused by these treatments to kill cancer cells. It also participates in the proliferation and creation of more malignant tumors. Furthermore, Rad51 is frequently over-expressed in cancer cells and its amount correlates in some way with tumor resistance to chemotherapy and the advancement of cancer [7], [8], [9], [10]. Inhibition of the cellular production of Rad51 by anti-sense and siRNA or ribozyme strategies slows down tumor development and increases the efficiency of anti-cancer treatments [11], [12], [13]. These data have revealed Rad51 as an attractive target for the development of inhibitors for anti-cancer therapies.

Aptamers are nucleic acid molecules that bind to a specific molecular target. These ligands are derived from combinatorial libraries through an in vitro selection strategy called Systematic Evolution of ligands by EXponential enrichment (SELEX) [14], [15]. They are structured single-stranded RNA or DNA, which bind tightly to the target molecules according to their 3-D structure. Oligonucleotide analogs can be employed, for example 2′-fluoro-pyrimidines, 2′-amino-pyrimidines, 2′-O-methyl pyrimidines, 2′-hydroxy-purines, and phosphorothioate [16]. These modifications can significantly increase aptamer functional half-life and thus in vivo exposure, especially when they are destined to have a potential therapeutic use. Thus, aptamers are particularly interesting molecules, able to bind to a variety of targets including proteins, peptides, enzymes, cell surface receptors, etc. [16]. They are considered to be good candidates in the development of new therapeutic agents [17], [18], [19].

In the work reported here, we applied the SELEX approach to HsRad51 and identified DNA aptamers capable of binding HsRad51 and inhibiting its strand exchange activity. We also showed their ability to affect strongly the HsRad51 filament formation on DNA, which is the first step of the strand exchange reaction. Selected aptamers were also shown to be specific towards HsRad51, as they did not interact with the prokaryotic ortholog RecA at low concentrations. These results reveal new analytical tools for research to study HR and open up new perspectives for the development of novel therapies targeting Rad51.

Section snippets

Proteins and peptides

The HsRad51 and EcRecA proteins were prepared as described previously [20], [21]. The peptide of 28 amino-acids derived from the BRC4 motif of BRCA2 protein (BRC4-28) was obtained from NeoSystem (France) [20].

Oligonucleotides

The oligonucleotide library for aptamer selection and the following oligonucleotides for the strand exchange reaction were obtained from MWG (Germany) and used without further purification. Aptamers and their fragments used for the biochemical studies were also chemically synthesized by

Selection of DNA aptamers directed against HsRad51

The selection of HsRad51-binding oligonucleotides was carried out using purified HsRad51 as the target protein and starting from an oligonucleotide library. As ATP is present in cells and required for HsRad51 activity, it was routinely added. The library was composed of oligonucleotides that are 94 nucleotides in length. They contained two fixed sequences of 22 nucleotides, one on each side of a central random domain of 50 nucleotides. Selection of oligonucleotides bound to HsRad51 was

Conclusion

By using the SELEX approach to target entire HsRad51, we selected three aptamers that inhibit its DNA strand exchange activity. These aptamers were able to disassemble the HsRad51/DNA filament and inhibit HsRad51 activity more efficiently than other inhibitors, such as the BRC4 peptide [20]. Furthermore, aptamers were shown to be selective for HsRad51. Finally, various experiments suggested that aptamers may interact with HsRad51 by their particular 3-D structure.

These observations support the

Acknowledgements

We thank Dr. Christophe Thiriet and Dr. Jullien Drone for valuable discussions and careful reading of the manuscript. The authors acknowledge the IMPACT Core facilities – Biogenouest. This work was supported by grants from the Association pour la Recherche sur le Cancer [grant number 3862] and the Region de Pays de la Loire [grant names CIMATH and MIAPS]. S.F.M. was a recipient of a PhD fellowship from CONICYT (Chile) and the French Embassy.

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