DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

Abstract

DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very lowfidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage Φ29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites.protein-primed DNA polymerase | Bam35 | abasic sites | translesion synthesis | isothermal DNA amplification R eplicative DNA polymerases (DNAPs) from A and B families, collectively termed replicases, exhibit a "tight fit" for their DNA and dNTP substrates and are wondrously adapted to form correct Watson-Crick base pairs, resulting in very pronounced fidelity (1, 2). This strict preference to produce A:T and G:C base pairs is also the Achilles heel of faithful DNA polymerases, however, because they are strongly inhibited by modified nucleotides present at sites of DNA damage, leading to the stalling of replication fork and eventually to replicative stress and cell death (3). At the stalled replication fork, the DNA polymerase may be exchanged by a translesion synthesis (TLS) polymerase, generally belonging to the Y family. These enzymes possess looser solventexposed active sites, which allows them to deal with aberrant DNA features much better, although with a very low polymerization accuracy with the risk of the accumulation of mutations and genetic instability (4, 5). Alternatively, nonbulky modified bases, such as uracil and 8-oxo-deoxyguanosine (8oxoG), can be bypassed by replicases, with faithful or mutagenic outcomes that can be modified by the sequence context and dNTP availability (6, 7).Abasic or apurinic/apyrimidinic (AP) sites are the most common DNA lesions arising in cells when the N-glycosydic bond between the sugar moiety and the nucleobase is broken, either spontaneously or by a DNA glycosylase reaction product in the base excision repair pathway (8, 9). Unrepaired abasic sites are highly blocking lesions for replicative DNA polymerases (10), although mutant polymerases with impaired proofreading activity or with mutations in the polymerization active site residues that affect the incoming nucleotide sel...

Plant organellar DNA polymerases paralogs exhibit dissimilar nucleotide incorporation fidelity

“…The method employed here to evaluate DNA fidelity is different from the method developed by Kunkel and coworkers . In this study, as in others, only the global error rate and not the error rate for each specific change can be detected . Despite these incompatibilities, we determined the frequency and identity of each change conferring the inactivation of the lacZ gene in pUC19 for each AtPolI as it has been described for other DNAPs .…”

“…In this study, as in others, only the global error rate and not the error rate for each specific change can be detected . Despite these incompatibilities, we determined the frequency and identity of each change conferring the inactivation of the lacZ gene in pUC19 for each AtPolI as it has been described for other DNAPs . Thus, we extracted plasmid DNA from independent white and light blue colonies from the lacZ fidelity assay and examined the nature and number of errors produced during MDA by sequencing 108 and 111 plasmids amplified with AtPolIA and AtPolIB, respectively (Fig.…”

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

“…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).…”

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

“…Proteinase K was from Affymetrix. Wild type Φ29DNAP, wild type Φ29TP and wild type Bam35 DNA polymerase (B35DNAP) were from the laboratory stocks and purified as described (29,35,36). …”

“…In addition to meeting these characteristics, Bam35 DNA polymerase (B35DNAP) is able to processively bypass abasic sites in DNA (29). These features, along with the presence of ITRs and a TP covalently linked to the viral genome (10), strongly suggest that Bam35 genome replication is performed by a protein-primed mechanism.…”