A precursor terminal protein-trinucleotide intermediate during initiation of adenovirus DNA replication: regeneration of molecular ends in vitro by a jumping back mechanism.
The adenovirus type 5 origin sequence starts with 3' GTAGTA. Initiation of replication occurs by a protein priming mechanism in which the viral precursor terminal protein (pTP) is covalently linked to the first nucleotide of the nascent chain, a dCMP residue. This suggests that a pTP-dCMP (pTP-C) complex functions as an initiation intermediate. Employing a reconstituted replication system and both synthetic oligonucleotides and the natural TP-DNA as templates, we show that pTP-CAT rather than pTP-C is an intermediate in initiation. By replicating oligonucleotide templates mutated at different positions and analyzing the product lengths, we observed that the GTA at positions 4-6, rather than 1-3, are used as a template for pTP-CAT formation. Moreover, deletions of one or two nucleotides at the molecular ends were regenerated upon in vitro replication. Our results support a model in which the pTP-CAT intermediate, synthesized opposite to positions 4-6, jumps back to position 1 of the template to start elongation. In order to permit elongation, some base pairing between pTP-CAT and template residues 1-3 is required. This jumping-back mechanism ensures the integrity of terminal sequences during replication of the linear genome.
King
A J
AJ
Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
van der Vliet
P C
PC
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017384:Sequence Deletion; D013698:Templates, Genetic
D004279:DNA, Viral; D015500:Chloramphenicol O-Acetyltransferase