Arabinofuranosyl nucleotides are not chain-terminators during initiation of new strands of DNA by DNA polymerase alpha-primase.
Polymerization of NTPs and arabinofuranosyladenosine triphosphate (araATP) during DNA polymerase alpha catalyzed elongation of primase-synthesized primers was examined. After primase synthesizes a primer, pol alpha normally polymerizes multiple dNTPs onto this primer. In the absence of a required dNTP, however, primers were still elongated by up to 35 nucleotides via polymerization of the corresponding NTP in place of the missing dNTP. During the elongation of exogenously added primer/templates, however, NTPs were not readily polymerized. AraATP was readily incorporated into products during elongation of primase-synthesized primers. Importantly, polymerization of araATP did not result in chain termination; rather, the next correct nucleotide was added such that araATP was simply an alternate substrate. In contrast, polymerization of araATP during elongation of exogenously added primer/templates resulted in strong chain termination. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer/templates with respect to interactions with dNTP analogs. Furthermore, these data provide a rationale for how araNMPs are efficiently incorporated into internucleotide linkages of DNA in whole cells and suggest that the initiation of new strands of DNA by pol alpha-primase may be a unique target for inhibiting replication.
Thompson
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HC
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Kuchta
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RD
D001088:Arabinonucleotides / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019915:DNA Primase; D004261:DNA Replication; D003854:Deoxyribonucleotides / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D001084:Vidarabine Phosphate / Q000031:analogs & derivatives / Q000378:metabolism
D001088:Arabinonucleotides; D003854:Deoxyribonucleotides; C034477:RNA primers; D001084:Vidarabine Phosphate; C000101:ara-ATP; D012313:RNA; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases