Search

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 ℙ29 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.

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3'-5' exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs

The N-glycosidic bond can be hydrolyzed spontaneously or by glycosylases during removal of damaged bases by the base excision repair pathway, leading to the formation of highly mutagenic apurinic/apyrimidinic (AP) sites. Organisms encode for evolutionarily conserved repair machinery, including specific AP endonucleases that cleave the DNA backbone 5′ to the AP site to prime further DNA repair synthesis. We report on the DNA polymerase X from the bacteriumBacillus subtilis (PolXBs) that, along with polymerization and 3′–5′-exonuclease activities, possesses an intrinsic AP-endonuclease activity. Both, AP-endonuclease and 3′–5′-exonuclease activities are genetically linked and governed by the same metal ligands located at the C-terminal polymerase and histidinol phosphatase domain of the polymerase. The different catalytic functions of PolXBs enable it to perform recognition and incision at an AP site and further restoration (repair) of the original nucleotide in a standalone AP-endonuclease-independent way.

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolXBs), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolXBs possesses an intrinsic 3’–5’ exonuclease activity specialized in resecting unannealed 3’-termini in a gapped DNA substrate. Biochemical analysis of a PolXBs deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/ archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3’–5’ exonuclease activity of PolXBs resides in its PHP domain. Furthermore, site-directed mutagenesis of PolXBs His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3’-termini resection by the 3’–5’ exonuclease activity of PolXBs in the DNA repair context are discussed.

To initiate phi 29 DNA replication, the DNA polymerase has to form a complex with the homologous primer terminal protein (TP) that further recognizes the replication origins of the homologous TP-DNA placed at both ends of the linear genome. By means of chimerical proteins, constructed by swapping the priming domain of the related phi 29 and GA-1 TPs, we show that DNA polymerase can form catalytically active heterodimers exclusively with that chimerical TP containing the N-terminal part of the homologous TP, suggesting that the interaction between the polymerase TPR-1 subdomain and the TP N-terminal part is the one mainly responsible for the specificity between both proteins. We also show that the TP N-terminal part assists the proper binding of the priming domain at the polymerase active site. Additionally, a chimerical phi 29 DNA polymerase containing the GA-1 TPR-1 subdomain could use GA-1 TP, but only in the presence of phi 29 TP-DNA as template, indicating that parental TP recognition is mainly accomplished by the DNA polymerase. The sequential events occurring during initiation of bacteriophage protein-primed DNA replication are proposed

The transcription factor Spo0A is a master regulator for entry into sporulation in Bacillus subtilis and also regulates expression of the virulent B. subtilis phage 29. Here, we describe a novel function for Spo0A, being an inhibitor of DNA replication of both, the 29 genome and the B. subtilis chromosome. Binding of Spo0A near the 29 DNA ends, constituting the two origins of replication of the linear 29 genome, prevents formation of 29 protein p6-nucleoprotein initiation complex resulting in inhibition of 29 DNA replication. At the B. subtilis oriC, binding of Spo0A to specific sequences, which mostly coincide with DnaA-binding sites, prevents open complex formation. Thus, by binding to the origins of replication, Spo0A prevents the initiation step of DNA replication of either genome. The implications of this novel role of Spo0A for phage 29 development and the bacterial chromosome replication during the onset of sporulation are discussed.

Phage ø29 is a virulent phage of Bacillus subtilis with no known lysogenic cycle. Indeed, lysis occurs rapidly following infection of vegetative cells. Here, we show that 29 possesses a powerful strategy that enables it to adapt its infection strategy to the physiological conditions of the infected host to optimize its survival and proliferation. Thus, the lytic cycle is suppressed when the infected cell has initiated the process of sporulation and the infecting phage genome is directed into the highly resistant spore to remain dormant until germination of the spore. We have also identified two host-encoded factors that are key players in this adaptive infection strategy. We present evidence that chromosome segregation protein Spo0J is involved in spore entrapment of the infected ø29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses ø29 development by repressing the main early ø29 promoters via different and novel mechanisms and also by preventing activation of the single late ø29 promoter.