Construct yeast cells carrying potential G4 forming sequences
Obtain NMR structures
Modify and develop new G4 ligands and perform biophysical tests using our fluorescence replacement assay
We observed genetic instability of certain G4 sequences carrying various internal size loops
Obtain the NMR structure of serval CEB25 G4s
obtain NMR structure of a G4 with PhenDC
Synthesized derivatives of existing ligands
Pursue the on going experiments
Prepare the methods of screening the yeast helices mutants and the collection of ligands
Manuscripts are planned for the end of 2013
DNA and RNA G-rich sequences are capable of forming secondary structures, known as G-quadruplexes (G4), corresponding to stacked coplanar guanines stabilized by Hoogsteen hydrogen bonding. G4 likely form from intra-molecular folding of single-stranded molecules containing at least four triplets of G, separated by a few nucleotides and can adopt a large variety of conformations, depending on the size and sequence of the intervening loops. Numerous sequences, potentially forming G4 structures are present in genomes but evidences concerning their in vivo formation and biological role(s) remains limitted.
We recently demonstrated in the yeast S. cerevisiae that, during leading strand replication, the G4-prone hCEB1 tandem array is destabilized upon treatment with the PhenDC3 G4 ligand and in the absence of the Pif1 helicase. Here, we propose to take advantage of our yeast system to address several novel issues.
First, determine the mechanisms of this replication-dependent instability. We will focus on understanding whether or not it depends on the formation of abnormally long single-stranded DNA regions prone to fold into G4, and determine the proteins involved in the folding and the maturation of these blocking structures; in particular we will address the redundant role of helicases and the bypass by the translesions polymerases.
Second, we wish to question whether the processing of G4 is mutagenic and determine the role of the G4s in promoting genome rearrangements, since G4 are often present in the vicinity of tumoral translocations.
Third, we wish to pursue a never reached structure/function analysis of few G-quadruplexes. A-T. Phan’s team (Singapore) have recently resolved the NMR G4 structure of our model human minisatellites hCEB1 and hCEB25. They form unexpected structures, different from each other and, in the case of CEB1, at least two different structures. We plan to construct a series of relevant CEB1 and CEB25 mutations (punctual single nucleotide changes, loop size and inter-repeat linkers variation, mixed motifs, etc…), in order to determine their NMR structure and assay their capacity to trigger instability.
Finally, as molecular G4 appear to differ one from another, and therefore, in principle may be differentially regulated and targeted by different drugs, we will use our yeast system to characterize the in vivo effects of series of new G4 ligands, developed by the chemistry lab of M-P.Teulade-Fichou (UMR 176, Institut Curie) and, in parallel, assay their potential to target other G4 sequences of therapeutic interest, such as the telomere repeats or the numerous potential G4s located in oncogene promoters.
Monsieur Alain NICOLAS (Institut Curie, Centre de Recherche) – email@example.com
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IC1 Institut Curie, Centre de Recherche
IC2 Institut Curie, Centre de Recherche
Help of the ANR 446,999 euros
Beginning and duration of the scientific project: October 2012 - 36 Months