Exploiting Pfu DNA polymerase's differential bypass capacity for AP vs. dU sites: a novel strategy for sensitive...

In biological systems, uracil repair in DNA is initiated by uracil-DNA glycosylase (UDG), which removes uracil bases from their corresponding sites to produce abasic sites (AP sites). This enzyme is conserved and functionally critical across living organisms, and its abnormal expression has been proven to be associated with some diseases. Both AP sites and uracil-containing sites (dU sites) impede the replication activity of Pfu DNA polymerase (Pfu-Pol), albeit through distinct mechanisms. AP sites, caused by base loss, generally slow down replication, while dU sites induce stalling by being recognized by the uracil-binding pocket of the enzymes. Our key finding demonstrates that the ability of Pfu-Pol to bypass AP sites is significantly greater than its capacity to traverse dU sites within an appropriate template concentration range, resulting in higher efficiency in generating full-length products. Exploiting this kinetic difference, we developed a simple and sensitive method for quantifying UDG activity. UDG converts dU-containing templates into AP-containing templates. This conversion accelerates Pfu-Pol-mediated amplification, which is quantitatively measured in a Quantitative Real-time PCR (qPCR) assay as a decrease in the cycle threshold (Ct) value. Under optimized conditions, the quantification of UDG was enabled within a range of 0.0001 to 0.01 U mL^-1 with high selectivity. UDG was successfully detected in Hela cell samples at protein inputs corresponding to 10 cells.