ena-STUDY-UMEA UNIVERSITY-17-04-2020-07:50:07:800-447
ChIP-seq of the catalytic subunit of the leading strand DNA polymerase (Cdc20) in the absence and presence of the G4 stabilizing drug PhenDC3
G-quadruplexes (G4) structures can be formed in nucleic acid stretches rich in Guanines. They are important for transcriptional and translational regulation, as well as for telomere maintenance. Unresolved G4 structures can induce replication stalling and genome instability. One way to study and detect G4 structures is to use substances or proteins that stabilizes or unwinds G4 structures. One such small stabilizing ligand that shows good affinity and selectivity for G4 structures is the bisquinolinium compound PhenDC3. The yeast strain S. pombe is a good model to study G4 structures because of its similar chromosomal features to higher eukaryotic organisms. It however has efficient efflux pumps making them resistant to many drugs. The Kapoor lab was previously able to engineer a S. pombe strain sensitive to drugs by deleting the two ABC transporter proteins, Pmd1 and Bfr1. In our present study, we show that this strain is also sensitive to PhenDC3 and make use of this to study which of the 446 bioinformatically predicted G4 sites in S. pombe that could impede replication. We accomplish this by using enrichment of the catalytic subunit of the leading strand DNA polymerase (Cdc20) as a marker for slowed down replication and perform ChIP-seq experiments of Cdc20 in the presence and absence of PhenDC3.
G-quadruplexes (G4) structures can be formed in nucleic acid stretches rich in Guanines. They are important for transcriptional and translational regulation, as well as for telomere maintenance. Unresolved G4 structures can induce replication stalling and genome instability. One way to study and detect G4 structures is to use substances or proteins that stabilizes or unwinds G4 structures. One such small stabilizing ligand that shows good affinity and selectivity for G4 structures is the bisquinolinium compound PhenDC3. The yeast strain S. pombe is a good model to study G4 structures because of its similar chromosomal features to higher eukaryotic organisms. It however has efficient efflux pumps making them resistant to many drugs. The Kapoor lab was previously able to engineer a S. pombe strain sensitive to drugs by deleting the two ABC transporter proteins, Pmd1 and Bfr1. In our present study, we show that this strain is also sensitive to PhenDC3 and make use of this to study which of the 446 bioinformatically predicted G4 sites in S. pombe that could impede replication. We accomplish this by using enrichment of the catalytic subunit of the leading strand DNA polymerase (Cdc20) as a marker for slowed down replication and perform ChIP-seq experiments of Cdc20 in the presence and absence of PhenDC3.