Telomeres are the nucleoprotein complexes that protect the end of eukaryotic chromosomes. In eukaryotes where telomeres are elongated by telomerase, the telomeric DNA strand running toward the 3' end (the G-strand) is generally composed of repeats of a short motif carrying consecutive guanines and ends with a 3' single-stranded overhang (the G-overhang). Because of the presence of consecutive guanines, telomeric G-strands are prone to fold into G-quadruplexes (G4) (Tran et al. 2011). G4 can form at telomeres in the single-stranded G-overhang as well as in the double-stranded region, notably during replication or transcription of TERRA (telomeric repeat-containing RNA) when the G-strand is transiently exposed as a single-strand.

Our main projects focus on the characterization of secondary structures formed by telomeric DNA and of their interaction with single-stranded DNA binding proteins involved in telomere metabolism, in particular the genome-wide RPA (Replication Protein A) and the shelterin POT1 (Protection of Telomere 1).

We have done ...

Structure of telomeric sequences. We characterised the structure and stability of long telomeric sequences in the presence of potassium (the physiological relevant cation for G4), demonstrating that they fold into contiguous G4 units non-interacting with each other and of similar stability (Bugaut et Alberti 2015). We also highlighted an original behaviour in sodium (Saintomé et al. 2016). These studies prompted us to investigate how human RPA and human POT1-TPP1 deal with these structures.

hRPA. After characterising the binding of hRPA to a single telomeric G4 (Salas et al. 2006, Safa et al. 2014, Safa et al. 2016), we demonstrated that hRPA efficiently binds to telomeric sequences structured into contiguous G4, unfolding them, independently of the number of G4 units (Lancrey et al. 2018). We also highlighted the potential of a dimeric G4 ligands to displace hRPA from telomeric sequences (Saintomé et al. 2018).

hPOT-TPP1. We provided evidence that multiple hPOT1-TPP1 bind in a cooperative mode proceeding from the 3' end toward 5', and unravelled the determinants of this behaviour (Chatain et al., under revision).

G4 are telomeres' best friends. We also studied an unstable variant motif, the GGGCTA motif, identified in the proximal ends of human telomeres. We showed that GGGCTA repeats can fold into hairpins poorly bound by hRPA, suggesting hypotheses about the length-dependent instability of GGGCTA arrays in human telomeres (Chatain et al. 2021). The GGGCTA telomeric variant motif (unstable, hairpin-prone and poorly bound by RPA in its hairpin form) acts as an enlightening counterpoint to GGGTTA telomeric canonical motif (stable, G4-prone and efficiently bound by RPA despite its structuring into contiguous G4) and raise original considerations about telomeric G4 and the evolution of telomeric motif.

We plan to do ...

Telomeres are a fascinating example of structural diversity across eukaryotes, built on a base of underlying unity. Our long-term goal is to work with RPA and POT1-like proteins from other eukaryotes with a double aim: (i) to search for hints of an evolution from a non-sequence specific SSB protein (RPA-like) toward a telomeric specific SSB protein (POT1-like), (ii) to investigate the structural features that allowed the coevolution of telomeric motifs and POT1-like proteins.

In the framework of a research project in collaboration with Stéphane Coulon (Centre de Recherche en Cancérologie de Marseille), Patrick Revy (Institut Imagine, Paris) and Caroline Kannengiesser (CHU Bichat), supported by an ANR funding, we are also working on variants of hRPA found in patients affected by idiopathic pulmonary fibrosis. We study whether these mutations affect the binding of hRPA to G4 structured telomeric sequences.

Our publications about telomeric G4 and/or RPA:

Chatain J, Blond A, Phan AT, Saintomé C, Alberti P. GGGCTA repeats can fold into hairpins poorly unfolded by Replication Protein A: a possible origin of the length-dependent instability of GGGCTA variant repeats in human telomeres. Nucleic Acids Res. 2021, doi.org/10.1093/nar/gkab518

Lancrey A, Safa L, Chatain J, Delagoutte E, Riou JF, Alberti P, Saintomé C. The binding efficiency of RPA to telomeric G-strands folded into contiguous G-quadruplexes is independent of the number of G4 units. Biochimie. 2018, doi: 10.1016/j.biochi.2017.11.017.

Saintomé C, Alberti P, Guinot N , Lejault P , Chatain J , Mailliet P , Riou JF , Bugaut A. Binding properties of mono- and dimeric pyridine dicarboxamide ligands to human telomeric higher-order G-quadruplex structures. Chem Commun. 2018, doi: 10.1039/c7cc07048a.

Safa L, Gueddouda NM, Thiébaut F, Delagoutte E, Petruseva I, Lavrik O, Mendoza O, Bourdoncle A, Alberti P, Riou JF, Saintomé C. 5' to 3' Unfolding Directionality of DNA Secondary Structures by Replication Protein A: G-quadruplexes and duplexes. J Biol Chem. 2016, doi: 10.1074/jbc.M115.709667.

Saintomé C, Amrane S, Mergny JL, Alberti P. The exception that confirms the rule: a higher-order telomeric G-quadruplex structure more stable in sodium than in potassium. Nucleic Acids Res. 2016, doi: 10.1093/nar/gkw003.

Bugaut A, Alberti P. Understanding the stability of DNA G-quadruplex units in long human telomeric strands. Biochimie. 2015, doi: 10.1016/j.biochi.2015.04.003.

Safa L, Delagoutte E, Petruseva I, Alberti P, Lavrik O, Riou JF, Saintomé C. Binding polarity of RPA to telomeric sequences and influence of G-quadruplex stability. Biochimie. 2014, doi: 10.1016/j.biochi.2014.04.006.

Tran PL, Mergny JL, Alberti P. Stability of telomeric G-quadruplexes. Nucleic Acids Res. 2011, doi: 10.1093/nar/gkq1292.

Salas TR, Petruseva I, Lavrik O, Saintomé C. Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA. Nucleic Acids Res. 2009, doi: 10.1093/nar/gkn895.

De Cian A, Grellier P, Mouray E, Depoix D, Bertrand H, Monchaud D, Teulade-Fichou MP, Mergny JL, Alberti P. Plasmodium telomeric sequences: structure, stability and quadruplex targeting by small compounds. Chembiochem. 2008, doi: 10.1002/cbic.200800330.

Salas TR, Petruseva I, Lavrik O, Bourdoncle A, Mergny JL, Favre A, Saintomé C. Human replication protein A unfolds telomeric G-quadruplexes. Nucleic Acids Res. 2006, doi: 10.1093/nar/gkl564.

Contributions and collaborations

We also contributed to several studies about G4 and/or RPA directed by other groups:

RPA in meiosis by G. Livera's team (DNA Repair 2021; doi: 10.1016/j.dnarep.2021.103097)

G4 embedded in a DNA duplex (a single-molecule study) by J.B. Boulé and V. Croquette's teams (Nucleic Acids Res. 2021, doi: 10.1093/nar/gkab306)

RPA in Arabidopsis by D. Shippen's team (Genetics 2020, doi: 10.1534/genetics.120.303222)

RPA-1/TEBP in Leishmania amazonensis by M.I.N. Cano's group (Biochim Biophys Acta Gen Subj. 2020, doi: 10.1016/j.bbagen.2020.129607)

G4 in Plasmodium spp genomes by J.J. Lopez-Rubio's team (PLoS Genet. 2020, doi: 10.1371/journal.pgen.1008917)

RPA and telomeres in S. pombe by S. Coulon's team (EMBO J. 2015,doi: 10.15252/embj.201490773)

G4 in replication origins by M.N. Prioleau's team (EMBO J. 2014, doi: 10.1002/embj.201387506)

G4 polymorphism in KiSS1 3' untranslated region by N. De Roux's team (Mol Cell Endocrinol. 2012, doi: 10.1016/j.mce.2011.12.014)

POT1 in WRN-deficient human cells by A. Londoño-Vallejo (Genes Dev. 2009, doi: 10.1101/gad.544009)