Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

Abstract

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in a number of linear genomes of viruses, linear plasmids and mobile elements. By this mechanism, a so-called terminal protein (TP) primes replication and becomes covalently linked to the genome ends. Bam35 belongs to a group of temperate tectiviruses infecting Gram-positive bacteria, predicted to replicate their genomes by a protein-primed mechanism. Here, we characterize Bam35 replication as an alternative model of protein-priming DNA replication. First, we analyze the role of the protein encoded by the ORF4 as the TP and characterize the replication mechanism of the viral genome (TP-DNA). Indeed, full-length Bam35 TP-DNA can be replicated using only the viral TP and DNA polymerase. We also show that DNA replication priming entails the TP deoxythymidylation at conserved tyrosine 194 and that this reaction is directed by the third base of the template strand. We have also identified the TP tyrosine 172 as an essential residue for the interaction with the viral DNA polymerase. Furthermore, the genetic information of the first nucleotides of the genome can be recovered by a novel single-nucleotide jumping-back mechanism. Given the similarities between genome inverted terminal repeats and the genes encoding the replication proteins, we propose that related tectivirus genomes can be replicated by a similar mechanism.

Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins

“…This mechanism is specific to linear genomes and has been well characterized in the podovirus ⌽29 (30, 31) but also in linear plasmids and other virus groups, including tectiviruses and adenoviruses (27,29,32,33). Protein-primed replication starts by the formation of a phosphoester bond, catalyzed by the viral DNA polymerase (DNAP), between the first nucleotide and the OH group of a serine, threonine, or tyrosine residue of a protein (the terminal protein [TP]) that acts as a primer and becomes covalently bound to both 5= ends of DNA (TP-DNA).…”

“…Finally, the lipid membrane encloses an approximately 15-kb linear double-stranded DNA genome with inverted terminal repeat sequences that is replicated by a proteinprimed mechanism (27)(28)(29). This mechanism is specific to linear genomes and has been well characterized in the podovirus ⌽29 (30, 31) but also in linear plasmids and other virus groups, including tectiviruses and adenoviruses (27,29,32,33).…”

“…Thus, the PRD1 initiation step involves the reiterative copy of the fourth base of a 3=-CCCC terminal repeat, and it requires that the initiation products [TP-(dGMP) n ] translocate backwards in three consecutive sliding-back steps to recover DNA end information (28). On the contrary, Bam35 initiation is directed by the third base of the 3=-ATA genome end, and a subsequent third-to-first template base singlenucleotide jumping-back process may be responsible for the recovery of the information of the 3= end sequence (29). Then, in both cases, the DNAP resumes the genome replication coupling strand displacement and processive DNA synthesis (29,34,35).…”

“…On the contrary, Bam35 initiation is directed by the third base of the 3=-ATA genome end, and a subsequent third-to-first template base singlenucleotide jumping-back process may be responsible for the recovery of the information of the 3= end sequence (29). Then, in both cases, the DNAP resumes the genome replication coupling strand displacement and processive DNA synthesis (29,34,35). Full-length TP-DNA replication can be achieved in vitro only with two proteins, namely, TP and DNAP (29,34,36).…”

“…Then, in both cases, the DNAP resumes the genome replication coupling strand displacement and processive DNA synthesis (29,34,35). Full-length TP-DNA replication can be achieved in vitro only with two proteins, namely, TP and DNAP (29,34,36). However, the presence of additional factors, like single-and double-stranded DNA-binding proteins, strongly stimulates the replication of PRD1 (37,38).…”