Polymerization of 2 '-fluoro-and 2 '-O-methyl-dNTPs by human DNA polymerase alpha, polymerase gamma, and primase

Studies were undertaken to assess the ability of human polymerase alpha (po l alpha) and polymerase gamma (pol gamma) 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, d UTP, UTP, and FIAUTP, in addition to normal deoxynucleotides. Pol alpha inc orporated all 2'-FdNTPs except 2'-FdATP, but not 2'-O-MedNTPs. Pol gamma in corporated all 2'-FdNTPs, but not 2'-O-MedNTPs. In general, 2'-fluorine sub stitution decreased V-max/K-m; however, the magnitude of the changes was nu cleotide dependent, with dATP and dUTP being the most affected. Misinsertio n frequencies for pol alpha and pol gamma of 2'-FdNTPs compared with their normal nucleotides were: FIAUTP > 2'-FdCTP > 2'-FdGTP > 2'-FdATP (pol gamma only) > 2'-FdUTP. Because kinetics of insertion of pol alpha can be affect ed by the nature of the primer, we examined the ability of pol alpha to pol ymerize 2'-fluoro- and 2'-O-MedATP and dGTP when elongating a primer synthe sized by DNA primase. Under these conditions, both 2'-FdATP and 2'-FdCTP we re polymerized, but 2'-O-MedATP and 2'-O-MedGTP were not. Primase alone cou ld 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 polymerase s can polymerize numerous 2'-FdNTPs but cannot polymerize 2'-O-MedNTPs. (C) 2000 Elsevier Science Inc.