DNA helicases play instrumental functions in DNA replication, transcription and repair. A number of missense mutations in human DNA helicase genes are linked to chromosomal instability diseases characterized by age-related disorders or associated with cancer [1]. Helicase inhibitors have the potential to impact several therapeutic areas, including oncology, viral and microbial diseases. Despite their extensive interest from the drug discovery industry, there are no approved drugs targeting helicases. To discover and successfully characterise new helicase inhibitors, effective high throughput enzyme assays are required. Critically, suitable mechanistic models of enzyme catalysis and function need to be developed. We here present a detailed description of the enzyme kinetics of a bacterial DNA helicase, using a differential equation-based model and a FRET-based assay approach.

The FRET assays were performed using as substrates ATP and a DNA forked duplex (tagged with a fluorophore and a non-emitting dark quencher). Using Origin C, a model employing ordinary differential equations was created specifically to detail this double-substrate enzyme reaction, considering a monomeric helicase forming a ternary complex with ATP and DNA, in which the initial DNA duplex can be spontaneously reformed in the assay solution.

Time course measurements were performed and the linearity of the initial rate was considered for the analysis of the double substrate titration. This last suggested the existence of an anti-cooperativity mechanism. Progress curves were obtained monitoring the enzyme activity in presence of different ATP and DNA combinations. The fit performed with the model created on Origin C described well the data and allowed us to calculate kinetic parameters like Km ATP, Km DNA and cooperativity. We measured the Km of ATP to be in the region of 10-100 µM at low DNA concentrations. Given previous HTS have been run at low DNA concentrations with high ATP concentrations, the conditions may not have been ideal to identify ATP competitive inhibitors.

DNA helicases catalytic mechanisms require a rationalisation that can be achieved with specific mathematical models. The description of such a kinetic enzyme mechanism will allow us to better understand inhibitory mechanisms to create more reliable helicase-targeting drugs.

[1] Brosh RM Jr (2013), DNA helicases involved in DNA repair and their roles in cancer. Nat. Rev. Cancer 13, 542–558.