family B-type DNA polymerase의 탈아미노산 된 염기의 sensing 기작연구

Abstract

The uracil-sensing domain in archaeal family B-type DNA polymerases recognizes the pro-mutagenic uracil in the DNA template, which leads to the stalling of DNA polymerases. Here, we describe our new findings regarding the recognition spectrum of the uracil-sensing domain of family B-type DNA polymerases, the molecular mechanism underpinning the stalling of polymerases, and a method to increase the efficiency of PCR amplification. The uracil-sensing domain of Pfu DNA polymerase appears to recognize hypoxanthine as well as uracil, as confirmed by primer extension assay, mutation study and molecular docking. Interestingly, we observed that two successive deaminated bases were required to stall TNA1 and KOD DNA polymerases while a single deaminated base was enough for stalling Pfu polymerase in spite of the virtually identical uracil-sensing domain. The facts that TNA1 and KOD DNA polymerases retain much higher extension rates than Pfu DNA polymerase and decrease of extension rate resulted in stalling TNA1 and KOD DNA polymerases at a single deaminated base, strongly suggest that the stalling is determined by the extension rate of DNA polymerases along with possessing uracil-sensing domain. It was demonstrated that dITP is spontaneously generated during PCR amplification and dITPase activity of HAM1-like protein from Thermococcus onnurineus NA1 enhances the PCR amplification yield of TNA1 and Pfu DNA polymerases.