title: Polymerization of 2'-fluoro- and 2'-O-methyl-dNTPs by human DNA polymerase alpha, polymerase gamma, and primase.
abstract: Studies were undertaken to assess the ability of human polymerase alpha (pol 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, dUTP, UTP, and FIAUTP, in addition to normal deoxynucleotides. Pol alpha incorporated all 2'-FdNTPs except 2'-FdATP, but not 2'-O-MedNTPs. Pol gamma incorporated all 2'-FdNTPs, but not 2'-O-MedNTPs. In general, 2'-fluorine substitution decreased V(max)/K(m) 2'-FdUTP. Because kinetics of insertion of pol alpha can be affected by the nature of the primer, we examined the ability of pol alpha 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.
authors.affiliation: NeXstar Pharmaceuticals, Inc., Boulder, CO 80301, USA. frichardson@nexstar.com
authors.forename: F C
authors.initials: FC
authors.lastname: Richardson
authors.forename: R D
authors.initials: RD
authors.lastname: Kuchta
authors.forename: A
authors.initials: A
authors.lastname: Mazurkiewicz
authors.forename: K A
authors.initials: KA
authors.lastname: Richardson
mesh_terms: D004256:DNA Polymerase I / Q000378:metabolism*; D000074002:DNA Polymerase gamma; D019915:DNA Primase / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D013942:Thymine Nucleotides / Q000737:chemistry / Q000378:metabolism; D014544:Uridine Triphosphate / Q000737:chemistry / Q000378:metabolism
chemical_list: D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; C029603:deoxyguanosine triphosphate; D019915:DNA Primase; D004256:DNA Polymerase I; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate; D014544:Uridine Triphosphate