Exploiting Pfu DNA Polymerase's differential bypass capacity for AP vs. deoxyuracil sites: a novel strategy for sensitive uracil-DNA glycosylase detection

Abstract

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 ezyme is conserved and functionally critical across living organisms, and its abnormal expression has been proved 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 binding to the enzyme's uracil-binding pocket. Our key finding demonstrates that Pfu-Pol's ability to bypass AP sites is significantly greater than its capacity to traverse dU sites, resulting in higher efficiency in generating full-length products. Leveraging 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 the 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 the optimized conditions, it enabled the quantification of UDG within a range of 0.0001 to 0.01 U/mL with high selectivity. UDG in Hela cell sample has been successfully detected with the usage of proteins equal to 10 Hela cells.