How cells deal with the inhibition of DNA replication initiation (14 min)
CDC7 links initiation and fork processing in DNA replication (12 min)
Our research aims to increase our knowledge of how the genome duplicates and use this information to develop novel therapeutic strategies against cancer. We have a keen interest in how genome stability is maintained during the cell cycle by the action of protein phosphorylation and ubiquitin mediated proteolysis.
A large body of our work focuses on defining the roles of the Cell Division Cycle 7 (CDC7) protein kinase and of its two regulatory subunits DBF4 and DRF1. Using genome editing we are investigating how each subunit directs the kinase to regulate essential aspects of DNA replication such as origin firing, replication fork stability and the DNA replication stress response.
Drugs inhibiting CDC7 are in development as novel anti-cancer agents and a greater understanding of CDC7 biology would greatly benefit the development of these novel targeted therapeutics. We are performing large-scale loss of function screens to identify genes and pathways important for cell survival and proliferation when CDC7 function is compromised, thus contributing to the development of CDC7 inhibition as a strategy for the treatment of cancer.
An artistic view of DNA replication intermediates visualised by DNA fibre technique and fluorescence microscopy.
Outline of genome-wide CRISPR/Cas9 loss of function screen
Cellular Responses to CDC7 Inhibition:
RIF1, ETAA1 and ATR: DNA replication initiates from multiple origins, and selective CDC7 kinase inhibitors (CDC7is) restrain cell proliferation by limiting origin firing. We have performed a CRISPR-Cas9 genome-wide screen to identify genes that, when lost, promote the proliferation of cells treated with sub-efficacious doses of a CDC7i. We have found that the loss of function of ETAA1, an ATR activator, and RIF1 reduce the sensitivity to CDC7is by allowing DNA synthesis to occur more efficiently, notably during late S phase. we also found that partial CDC7 inhibition induces ATR mainly through ETAA1, and that if ATR is subsequently inhibited, origin firing is unleashed in a CDK- and CDC7-dependent manner. Cells are then driven into a premature and highly defective mitosis, a phenotype that can be recapitulated by co-depletion of the two ATR activators: ETAA1 and TOPBP1.
PTPB1: in the same CRISPR-Cas9 screen we identified the Polypyrimidine Tract Binding Protein 1 (PTBP1) as an important factor in the response to CDC7 inhibition. Compromised PTBP1 expression makes cells defective in RPA recruitment, genomically unstable, and resistant to CDC7 inhibitors. PTBP1 deficiency hampers ATR activation by affecting the expression and splicing of many genes. We find that an exon skipping event in RAD51AP1 contributes to checkpoint deficiency in PTBP1-deficient cells. These results identify PTBP1 as a key factor in replication stress response and define how ATR activity modulates the activity of CDC7 inhibitors.
A New Function of CDC7 at Stalled Replication Forks:
During elongation, ongoing replication forks (Play) encounter obstacles causing them to arrest (Pause) and regress (Rewind) into a four-branched structure. These remodelled forks require controlled nucleolytic processing in order to restart (Resume) DNA synthesis.
We have found that CDC7 kinase acts not only at replication origins but also at paused forks where it coordinates MRE11-dependent fork processing, contributing to fork restart and modulating fork speed. Upon prolonged arrest (Stop) and compromised fork protection (e.g. BRCA2 defects), CDC7 promotes fork degradation contributing to replication dependent chromosome breakage.
Mechanism of Action of CDC7 Inhibitors:
The CDC7 kinase, by phosphorylating the MCM DNA helicase, is a key switch for DNA replication initiation. ATP competitive CDC7 inhibitors are being developed as potential anticancer agents. We have characterized the mode of action of the two widely used CDC7 inhibitors, which have become important tool compounds to explore the kinase’s cellular functions and we have used a chemical genetics approach to further characterize pharmacological CDC7 inhibition. We have found that in human breast cells, CDC7 is essential and that CDC7 kinase activity is formally required for proliferation. However, full and sustained inhibition of the kinase, which is required to block the cell-cycle progression with ATP competitive inhibitors, is problematic to achieve. We established that MCM2 phosphorylation is highly sensitive to CDC7 inhibition and, as a biomarker, it lacks in dynamic range since it is easily lost at concentrations of inhibitors that only mildly affect DNA synthesis. Furthermore, we found that the cellular effects of selective CDC7 inhibitors can be altered by the concomitant inhibition of cell-cycle and transcriptional CDKs.
Full story at https://pubs.acs.org/doi/10.1021/acschembio.7b00117
USP9x in Genome Stability Maintenance:
The ubiquitin-specific protease 9X (USP9X) contributes to genome stability during DNA replication and chromosome segregation. Depletion of USP9X leads to DNA double-strand breaks, some of which are triggered by replication fork collapse. We have identified CLASPIN, which mediates multiple protein-protein interactions at the replication fork, as a key target of USP9X in S-phase.
More recently we discovered that USP9X can be considered as a novel regulator of homologous recombination (HR) DNA repair in human cells. By performing cellular HR reporter, irradiation-induced focus formation and colony formation assays, we have shown that USP9X is required for efficient HR. Mechanistically USP9X is important to sustain the expression levels of key HR factors, namely BRCA1 and RAD51 through a non-canonical regulation of their mRNA abundance. Intriguingly, we find that the contribution of USP9X to BRCA1 and RAD51 expression is independent of its known catalytic activity.
More on USP9X in DNA replication at : https://cancerres.aacrjournals.org/content/76/8/2384
and USP9x in DNA repair at: https://jcs.biologists.org/content/133/3/jcs233437X
DNA Mediated Chromatin Pull Down
Our first Galway PhD student established a technique for the purification and characterization of proteins associated to newly replicated DNA that we called DNA mediated chromatin pull down (Dm-ChP). Over the years, this technique has been particularly useful in understanding which proteins acts at forks during unperturbed replication, upon replication stress and in different genetic backgrounds.
See the original at: https://www.nature.com/articles/srep00095