Polymerization of 2′-fluoro- and 2′- O-methyl-dNTPs by human DNA polymerase α, polymerase γ, and primase

Studies were undertaken to assess the ability of human polymerase α (pol α) and polymerase γ (pol γ) to incorporate 2′-fluoro- and 2′- O-methyldeoxynucleotides into DNA. In vitro DNA synthesis systems were used to detect incorporation and determine K m and V max for 2′-FdATP, 2′-FdUTP, 2′-FdCTP, 2′-FdGTP, 2′- O-MedATP, 2′- O-MedCTP, 2′- O-MedGTP, 2′- O-MedUTP, dUTP, UTP, and FIAUTP, in addition to normal deoxynucleotides. Pol α incorporated all 2′-FdNTPs except 2′-FdATP, but not 2′- O-MedNTPs. Pol γ incorporated all 2′-FdNTPs, but not 2′- O-MedNTPs. In general, 2′-fluorine substitution decreased V max /K m ; however, the magnitude of the changes was nucleotide dependent, with dATP and dUTP being the most affected. Misinsertion frequencies for pol α and pol γ of 2′-FdNTPs compared with their normal nucleotides were: FIAUTP > 2′-FdCTP > 2′-FdGTP > 2′-FdATP (pol γ only) > 2′-FdUTP. Because kinetics of insertion of pol α can be affected by the nature of the primer, we examined the ability of pol α to polymerize 2′-fluoro- and 2′- O-MedATP and dGTP when elongating a primer synthesized by DNA primase. Under these conditions, both 2′-FdATP and 2′-FdGTP were polymerized, but 2′- O-MedATP and 2′- O-MedGTP were not. Primase alone could not readily polymerize these analogs into RNA primers. Previous studies showed that 2′-deoxy-2′-fluorocytosine (2′-FdC) is incorporated by several non-human DNA polymerases. The current studies showed that human polymerases can polymerize numerous 2′-FdNTPs but cannot polymerize 2′- O-MedNTPs.