Cell experiment:

Briefly, untreated WT cells and pif1Δ cells from a fresh patch of cells get from a single colony bearing the parental allele size are diluted in 5 mL of YPD (2×105 cells/mL), grown for 8 generations at 30℃ with shaking, and spreaded as single colony on YPD plates. To measure minisatellite instability upon Phen-DC3 Trifluoromethanesulfonate treatment, WT cells from a fresh patch on YPD are grown for 8 generations at 30℃ in liquid SC containing Phen-DC3 Trifluoromethanesulfonate at 10 μM. Isolated colonies or pools of colonies are analyzed by Southern blot using the EcoRI digestion that cuts at each side of the minisatellite[2].

Phen-DC3 Trifluoromethanesulfonate is a G-quadruplex (G4) specific ligand which can inhibit FANCJ and DinG helicases with IC50s of 65±6 and 50±10 nM, respectively.

In WT cells, a CEB1-WT array is rather stable but undergoes frequent rearrangements upon addition of 10 μM Phen-DC3 Trifluoromethanesulfonate (Phen-DC3). It is found that the c-Myc allele exhibits significant destabilization upon Phen-DC3 Trifluoromethanesulfonate treatment and PIF1 deletion. The CEB25-L111(T) array is stable in WT cells, it becomes unstable upon addition of Phen-DC3 Trifluoromethanesulfonate or deletion of PIF1. It is also highly destabilized in the presence of Phen-DC3 Trifluoromethanesulfonate or in the absence of PIF1. The CEB1-loop CEB25 allele remaines fully stable in both PIF1-treated and WT cells[2].

[1]. Sanjay Kumar Bharti, et al. Specialization among Iron-Sulfur Cluster Helicases to Resolve G-quadruplex DNA Structures That Threaten Genomic Stability. J Biol Chem. 2013 Sep 27; 288(39): 28217-28229. [2]. Aurèle Piazza, et al. Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites. EMBO J. 2015 Jun 12; 34(12): 1718-1734.