Phage φ29 and Nf terminal protein-priming domain specifies the internal template nucleotide to initiate DNA replication

Abstract

Bacteriophages 29 and Nf from Bacillus subtilis start replication of their linear genome at both DNA ends by a protein-primed mechanism, by which the DNA polymerase, in a template-instructed reaction, adds 5 -dAMP to a molecule of terminal protein (TP) to form the initiation product TP-dAMP. Mutational analysis of the 3 terminal thymines of the Nf DNA end indicated that initiation of Nf DNA replication is directed by the third thymine on the template, the recovery of the 2 terminal nucleotides mainly occurring by a stepwise sliding-back mechanism. By using chimerical TPs, constructed by swapping the priming domain of the related 29 and Nf proteins, we show that this domain is the main structural determinant that dictates the internal 3 nucleotide used as template during initiation.chimerical terminal protein ͉ polymerase ͉ linear genomes ͉ protein-primed replication ͉ sliding-back M any organisms, such as bacteriophages; animal viruses, such as adenovirus, mitochondrial plasmids, linear chromosomes and plasmids of Streptomyces (1); and more recently virus infecting Archaea, such as halovirus (2, 3), possess replication origins constituted by inverted terminal repetitions (ITR) with a terminal protein (TP) linked to both 5Ј ends of their linear chromosomes (4). In these cases, the location of the 2 replication origins allows both strands to be replicated continuously, without requiring asymmetric complexes of DNA polymerase with other accessory proteins to control the different mechanics of continuous and discontinuous DNA synthesis (5). Additionally, the TP provides the OH Ϫ group of a specific serine, threonine or tyrosine to prime initiation of DNA replication from the very ends of the linear chromosome, the TP remaining covalently linked to the 5Ј-DNA ends (parental TP) (1,4,6).The development of an in vitro replication system with highly purified proteins and DNA from bacteriophage 29 of B. subtilis has allowed to lay the foundations of the so-called proteinprimed mechanism of DNA replication (4, 6). 29 has a linear dsDNA, 19,285 bp long, containing a TP of 31 kDa covalently linked to each 5Ј end [TP-DNA (7)] that, together with a 6-bp inverted terminal repeat (3Ј-TTTCAT-5Ј) (8, 9) form part of a minimal replication origin. Once the replication origins are specifically recognized by a heterodimer formed by the DNA polymerase and a free TP molecule (10, 11), the DNA polymerase catalyzes the formation of a covalent bond between dAMP and the hydroxyl group of Ser 232 of the TP, a reaction directed by the second T at the 3Ј end (12). Then, the TP-dAMP initiation product translocates backwards 1 position to recover the template information corresponding to the first T, the so-called sliding-back mechanism, which requires a terminal repetition of 2 bp (12) and provides a way to prevent mutations at the 29 DNA ends during initiation, since the 3Ј-5Ј exonuclease activity of 29 DNA polymerase cannot proofread the TP-linked nucleotide (13).The sliding-back mechanism, occurring also in the 29-related phage GA-1 (14); S...

Telomere associated primase Tap repairs truncated telomeres of Streptomyces

“…The Φ29-related phage Nf and the Streptococcus pneumoniae phage Cp-1 initiate replication at the 3΄ third nucleotide of the template ( 3΄ TTT) (23,24). E. coli phage PRD1 initiates at the 3΄ fourth nucleotide ( 3΄ CCCC) (25).…”

Insights into the Determination of the Templating Nucleotide at the Initiation of φ29 DNA Replication

“…Previous assays showed that the priming domain is the main structural determinant that dictates the internal 3Ј nucleotide used as template during initiation (21). Here, we have analyzed the role of the aromatic residue Phe-230 of the TP priming loop, proximal to the priming Ser-232.…”

“…assays with chimerical TPs indicated that the priming domain is responsible for placing the templating nucleotide at the polymerization active site during initiation (21). The TP priming domain (residues 173-266) is comprised of a four-helix bundle structure, the priming loop (residues 227-233) being located between the last and penultimate ␣ helixes of the domain (19).…”

“…Previous studies performed with chimerical TPs allowed to propose that the priming domain is responsible for placing the internal 3Ј nucleotide used as template during initiation at the polymerization active site (21). The results presented in this paper indicate that although the parental TP is not playing a significant role in the recognition of the template nucleotide used to direct the initiation reaction in 29 TP-DNA replication, the correct positioning of the 3Ј terminus of the template strand at the polymerase active site relies on residue Phe-230 of the TP priming loop, allowing the penultimate T to direct the insertion of the initiator dAMP.…”

Multiple roles of genome-attached bacteriophage terminal proteins

“…the priming loop (Longás et al, 2008). Then, after a transition step, the DNAP dissociates, and the elongation continues until replication of the nascent DNA strand is completed, thanks to the high processivity of the DNAP and its intrinsic strand displacement activity.…”