Enzolution(R) WRN Helicase TR-FRET Assay System

The Enzolution(R) WRN Helicase Assay System is intended for use with the Transcreener(R) ADP2 Assay Kits to measure enzymatic activity for WRN Helicase (Also known as Werner Syndrome ATP-dependent Helicase). WRN is a multifunctional enzyme that has both magnesium and ATP-dependent DNA-helicase activity and 3'->5' exonuclease activity towards double-stranded DNA with a 5'-overhang. ADP formation by the WRN helicase can be detected using the Transcreener(R) ADP2 assay, a far-red, competitive fluorescence assay that enables single addition, mix-and-read detection in a continuous or endpoint format. The assay has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers. The WRN Helicase Assay System provides all reagents required to screen and profile WRN Helicase inhibitors when used with the Transcreener(R) ADP2 Assay Kits, which are available with FP, FI and TR-FRET readouts. The protocol is configured for 384-well plates, use of different multi-well plate formats will require adjustment of reagent concentrations utilized in the assay. Key Applications:  Screening for WRN Helicase inhibitors (or activators)  Generating dose response curves and IC50 values for WRN Helicase inhibitors  Kinetic and mechanistic analyses Protein Function: Multifunctional enzyme that has magnesium and ATP-dependent 3'-5' DNA-helicase activity on partially duplex substrates (PubMed:9224595, PubMed:9288107, PubMed:9611231). Also has 3'->5' exonuclease activity towards double-stranded (ds)DNA with a 5'-overhang (PubMed:11863428). Has no nuclease activity towards single-stranded (ss)DNA or blunt-ended dsDNA (PubMed:11863428). Helicase activity is most efficient with (d)ATP, but (d)CTP will substitute with reduced efficiency, strand displacement is enhanced by single-strand binding-protein (heterotrimeric replication protein A complex, RPA1, RPA2, RPA3) (PubMed:9611231). Binds preferentially to DNA substrates containing alternate secondary structures, such as replication forks and Holliday junctions. May play an important role in the dissociation of joint DNA molecules that can arise as products of homologous recombination, at stalled replication forks or during DNA repair. Alleviates stalling of DNA polymerases at the site of DNA lesions. Plays a role in the formation of DNA replication focal centers, stably associates with foci elements generating binding sites for RP-A (By similarity). Plays a role in double-strand break repair after gamma-irradiation (PubMed:9224595, PubMed:9288107, PubMed:9611231). Unwinds some G-quadruplex DNA (d(CGG)n tracts), unwinding seems to occur in both 5'-3' and 3'-5' direction and requires a short single-stranded tail (PubMed:10212265). d(CGG)n tracts have a propensity to assemble into tetraplex structures, other G-rich substrates from a telomeric or IgG switch sequence are not unwound (PubMed:10212265). Depletion leads to chromosomal breaks and genome instability (PubMed:33199508) [The Uniprot Consortium]