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A precursor terminal protein‐trinucleotide intermediate during initiation of adenovirus DNA replication: regeneration of molecular ends in vitro by a jumping back mechanism.
Author(s) -
King A.J.,
Vliet P.C.
Publication year - 1994
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1994.tb06917.x
Subject(s) - biology , dna replication , genetics , dna , posttranslational modification , in vitro , biochemistry , enzyme
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.