5′ to 3′ Unfolding Directionality of DNA Secondary Structures by Replication Protein A G-QUADRUPLEXES AND DUPLEXES

Layal Safa; Nassima Meriem Gueddouda; Frédéric Thiébaut; Emmanuelle Delagoutte; Irina Petruseva; Olga Lavrik; Oscar Mendoza; Anne Bourdoncle; Patrizia Alberti; Jean-François Riou; Carole Saintomé

doi:10.1074/jbc.M115.709667

5′ to 3′ Unfolding Directionality of DNA Secondary Structures by Replication Protein A

G-QUADRUPLEXES AND DUPLEXES*

From the ^‡Structure et Instabilité des Génomes, Sorbonne Universités, Muséum National d'Histoire Naturelle, INSERM U1154, CNRS UMR 7196, CP26, 57 rue Cuvier, 75005, Paris, France,
the ^§Sorbonne Universités, UPMC University Paris 06, F-75005, Paris, France,
the ^¶Laboratoire ARNA-INSERM U1212, UMR 5320, Institut européen de chimie et biologie, 2 rue Robert Escarpit, 33607 Pessac, France,
the ^‖Ecole Normale Supérieure, PSL Research University, Département de Chimie, 24 rue Lhomond, CNRS, UMR 7203 LBM, 75005 Paris, France, and
the ^**Novosibirsk Institute of Chemical Biology and Fundamental Medecine, Siberian Division of Russian Academy of Science, 630090 Novosibirsk, Russia

↵3 To whom correspondence may be addressed. E-mail: alberti{at}mnhn.fr.
↵4 To whom correspondence may be addressed: Muséum National d'Histoire Naturelle, INSERM U1154, CNRS UMR 7196, F-75005, Paris, France. Tel.: 33-140793686; Fax: 33-1-40-79-37-05; E-mail: carole.saintome{at}mnhn.fr.

Abstract

The replication protein A (RPA) is a single-stranded DNA-binding protein that plays an essential role in DNA metabolism. RPA is able to unfold G-quadruplex (G4) structures formed by telomeric DNA sequences, a function important for telomere maintenance. To elucidate the mechanism through which RPA unfolds telomeric G4s, we studied its interaction with oligonucleotides that adopt a G4 structure extended with a single-stranded tail on either side of the G4. Binding and unfolding was characterized using several biochemical and biophysical approaches and in the presence of specific G4 ligands, such as telomestatin and 360A. Our data show that RPA can bind on each side of the G4 but it unwinds the G4 only from 5′ toward 3′. We explain the 5′ to 3′ unfolding directionality in terms of the 5′ to 3′ oriented laying out of hRPA subunits along single-stranded DNA. Furthermore, we demonstrate by kinetics experiments that RPA proceeds with the same directionality for duplex unfolding.

Footnotes

↵1 Supported by a doctoral fellowship from the “Ligue Nationale contre le Cancer.”
↵2 Supported by a doctoral fellowship from the “Programme Doctoral Interfaces pour le Vivant 2014, Sorbonne Universités, UPMC Univ Paris 06.”
↵* This work was supported in part by grants from the “Ligue Nationale contre le Cancer, Equipe Labelisée 2010,” Agence Nationale de la Recherche Grant ANR-09-BLAN-0355, Institut National du Cancer Grant TP53-INTRON3 (to J. F. R., E. D., P. A., and C. S.), and a grant from the Program on Molecular Cellular Biology RAS (to O. L.). The authors declare that they have no conflicts of interest with the contents of this article.