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Helicase enzymes use the energy from nucleotide triphosphate (NTP) hydrolysis 1 to resolve DNA secondary structures generating single-stranded DNA 2. PIF1 is a 5'–3' helicase belonging to SF1B (Super Family 1B) helicase family, first identified in yeast and then in bacterial and human cells. Human PIF1 (hPIF1) plays roles in genome maintenance; in vitro, it has double-stranded DNA (dsDNA) and G quadruplex (G4) DNA unwinding activity and G4 DNA specific binding activity 2. It recognizes and processes synthetic stalled DNA replication fork-like substrates reversing the replication strand to restart replication 3. In intact cells, hPIF1 is required for replication fork stability; some tumour cells depend upon hPIF1 and siRNA-mediated knockdown causes their apoptosis, suggesting potential therapeutic applications of PIF1.
In this work, hPIF1 and unimolecular G4 DNA interaction have been investigated through an internally controlled ‘pull-down’ DNA binding assay. Several unimolecular G4 DNA substrates with different structures and conformations were tested; changes in apparent G4 DNA binding affinity during the ATP hydrolysis cycle were investigated by reproducing ATP hydrolysis steps with nucleotide cofactor analogues.
In G4 DNA unwinding assay hydrolyzable ATP is not essential while ADP·MgF4- is most efficient at stimulating unwinding. The data suggest that G4 DNA is recognised in the apo state, ATP hydrolysis leads to structural rearrangements and an increase in ssDNA binding affinity, presumably linked to translocation and G4 DNA unwinding. To identify hPIF1 residues involved in G4 DNA interaction, a model based on ToPIF1- G4 DNA crystal structure was tested by biochemical assays. However preliminary data suggested that the binding model may not be universal.
A second branch of this work is focused on the discovery of hPIF1 inhibitors. A collaboration with Edelris Keymical (Lyon, France) followed three different approaches for novel small molecule inhibitors identification, resulting in new compounds able to inhibit hPIF1 helicase activity.