The adenovirus priming protein pTP contributes to the kinetics of initiation of DNA replication.
Adenovirus (Ad) precursor terminal protein (pTP) in a complex with Ad DNA polymerase (pol) serves as a primer for Ad DNA replication. During initiation, pol covalently couples the first dCTP with Ser-580 of pTP. By using an in vitro reconstituted replication system comprised of purified proteins, we demonstrate that the conserved Asp-578 and Asp-582 residues of pTP, located close to Ser-580, are important for the initiation activity of the pTP/pol complex. In particular, the negative charge of Asp-578 is essential for this process. The introduced pTP mutations do not alter the binding capacity to DNA or polymerase, suggesting that the priming mechanism is affected. The Asp-578 or Asp-582 mutations increase the Km for dCTP incorporation, and higher dCTP concentrations or Mn2+ replacing Mg2+ partially relieve the initiation defect. Moreover, the kcat/Km values are reduced as a consequence of the pTP mutations. These observations demonstrate that pTP influences the catalytic activity of pol in initiation. Since both Asp residues are situated close to the pol active site during initiation, they may contribute to correct positioning of the OH group in Ser-580. Our results indicate that specific amino acids of the protein primer influence the ability of Ad5 DNA polymerase to initiate DNA replication.
Department of Physiological Chemistry, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
Monika E
ME
Mysiak
P Elly
PE
Holthuizen
Peter C
PC
van der Vliet
D000260:Adenoviruses, Human / Q000235:genetics*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry; D014764:Viral Proteins; D014779:Virus Replication
D004279:DNA, Viral; D010750:Phosphoproteins; D011498:Protein Precursors; D014764:Viral Proteins; C085476:pTP protein, adenovirus; D001224:Aspartic Acid; D008345:Manganese; D012694:Serine; D004259:DNA-Directed DNA Polymerase