WP2
(I) Engineered human cell lines with programmable replication fork barriers
The replication fork barrier is based on the LacO/LacR system of Escherichia coli. By using the CRISPR-Cas9 knock-in technology a LacO array cassette was inserted into human Chromosome 1 in U20S cells to create a replication fork barrier that can be switched on by expressing the ER-LacR-mCherry fusion protein via simple plasmid transfection. This system can be used to analyse the effects of downregulating a DNA helicase of interest on the ability of the replication machinery to overcome the above protein-DNA obstacles.
(II) Human cell lines where specific DNA helicases are knocked-down/-out
The following human cell lines were generated where specific DNA helicases are knocked-down/-out (KD/KO): 1) U2OS and RPE cell lines, where BLM is KO; 2) U2OS cell lines, where RTEL1 is KD by siRNAs and U2OS lines complemented with RTEL1 wild type and the helicase-dead variant (K48R); 3) FANCJ-KO HeLa cell lines complemented with FANCJ wild type or FANCJ AALA mutant.
WP3
(I) DDX11 and FANCJ
Direct interactions of DDX11 and FANCJ with the DNA replication factories were discovered at CNR (Naples, Italy). DDX11-binding residues of Timeless were identified. A FANCJ site-specific mutant (named FANCJ AALA) unable to bind the DNA replication machinery was discovered and used to demonstrate that association of FANCJ to the ongoing replication forks is critical to counteract replication stress.
(II) DHX30
Studies carried out at UCPH (Copenhagen, Denmark) revealed that the RNA helicase DHX30 is associated with the subunit 3 of DNA polymerase delta (PolD3) in human cells. DHX30 depletion was found to induce increased mitotic DNA synthesis (MiDAS) in several cell lines at telomeres and common fragile sites. Biochemical analysis of various human DHX30 isoforms is under way.
(III) PIF1
Site-specific mutagenesis studies of human PIF1 led to the identification of residues potentially involved in binding G4 DNA structures in the laboratory of USFD (Sheffield, United Kingdom).
(IV) RTEL1
One important task, accomplished at ELETTRA (Trieste, Italy), was the production in soluble and catalytically active form of the Caenorhabditis elegans (Ce) and human RTEL1 DNA helicases in Escherichia coli cells.
WP4 - Biophysical analyses at single-molecule level
(I) The BLM complex and PICH
Single-molecule biophysical studies were carried out to investigate the action mechanism of PICH, a DNA translocase, which interacts with the BLM complex and plays an important role in resolving the peculiar cytological structures known as “ultrafine anaphase bridges” (UFBs). Using the C-Trap system (Lumicks BV) a DNA loop extrusion capability of PICH was visualised at UCPH (Copenhagen, Denmark).
WP5 - Drug discovery and design
(I) BLM
A high throughput screening (HTS) of a small molecule compound library (333440 compounds) was carried out at LDC (Dortmund, Germany) to identify inhibitors of the BLM helicase using a miniaturised fluorescence-based DNA unwinding assay. 83 compounds were identified that inhibit BLM, but on the RECQ1 DNA helicase.
(II) RECQ1
Identification of RECQ1 helicase inhibitors was based on either computational methods or a HTS using a LDC compound library subset (16000 molecules). A total of 43 compounds derived from the virtual screening were also present in the hit list derived from the HTS. In particular, the compound LDC210712, expected to target the RECQ1 DNA binding site, was listed as a top virtual hit and validated as a true RECQ1 inhibitor.
(III) PIF1
To identify human PIF1 inhibitors a structure-based drug design approach was used, since the X-ray structure of this protein is available. Docking of 10098 compounds from Edelris’ Keymical CollectionsTM led to the identification of 96 compounds potentially binding to a PIF1 pocket, adjacent to the single-strand DNA binding site. Among them, 3 compounds (named A6, B6, C6), which share a common core chemical structure, were found to inhibit the PIF1 DNA helicase activity in vitro. These studies were done by EDELRIS (Lyon, France) and USFD (Sheffield, United Kingdom).
WP6
Bioinformatic analyses were carried out at UKESSEN (Essen, Germany) revealed that: 1) DNA helicases are over-expressed in most tumour samples compared to matched healthy tissues; 2) in most cases, high or low level of expression of the target DNA helicases correlates with poor or higher patients’ survival, respectively; 3) in melanoma entities high expression of FANCJ/BRIP1 is connected with poor prognosis; while, low expression of RECQ4 associates with higher survival rate.
