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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.

Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage 29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.

Uracil-DNA glycosylase (UDG) is a conserved DNA repair enzyme involved in uracil excision from DNA. Here, we report the biochemical characterization of UDG encoded by Bacillus subtilis, a model low G+C Gram-positive organism. The purified enzyme removes uracil preferentially from single-stranded DNA over double-stranded DNA, exhibiting higher preference for U:G than U:A mismatches. Furthermore, we have identified key amino acids necessary for B. subtilis UDG activity. Our results showed that Asp-65 and His-187 are catalytic residues involved in glycosidic bond cleavage, whereas Phe-78 would participate in DNA recognition. Recently, it has been reported that B. subtilis phage φ29 encodes an inhibitor of the UDG enzyme, named protein p56, whose role has been proposed to ensure an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracils present in the φ29 genome. In this work, we also show that a φ29-related phage, GA-1, encodes a p56-like protein with UDG inhibition activity. In addition, mutagenesis analysis revealed that residue Phe-191 of B. subtilis UDG is critical for the interaction with φ29 and GA-1 p56 proteins, suggesting that both proteins have similar mechanism of inhibition.

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., Spanish Ministry of Education and Science (BFU 2005- 00733 and Consolider-Ingenio 2010 24717) to M.S.; Fundacio´ n Ramo´ n Areces to the Centro de Biologı´ a Molecular ‘Severo Ochoa’. Funding for open access charge: Spanish Ministry of Education and Science (Consolider-Ingenio 2010 24717).

Prokaryotic DNA replication is compartmentalized at the cellular membrane. The Bacillus subtilis phage φ29-encoded membrane protein p16.7 is one of the few proteins known to be involved in the organization of prokaryotic membrane-associated DNA replication. The functional DNA binding domain of p16.7 is constituted by its C-terminal half, p16.7C, which forms high affinity dimers in solution and which can form higher order oligomers. Recently, the solution and crystal structures of p16.7C and the crystal structure of the p16.7C-DNA complex have been solved. Here, we have studied the p16.7C dimerization process and the structural and functional roles of p16.7 residues Trp-116 and Asn-120 and its last nine C-terminal amino acids, which form an extended tail. The results obtained show that transition of folded dimers into unfolded monomers occurs without stable intermediates and that both Trp-116 and the C-terminal tail are important for dimerization and functionality of p16.7C. Residue Trp-116 is involved in formation of a novel aromatic cage dimerization motif, which we call “Pro cage.” Finally, whereas residue Asn-120 plays a minor role in p16.7C dimerization, we show that it is critical for both oligomerization and DNA binding, providing further evidence that DNA binding and oligomerization of p16.7C are coupled processes.

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., Funding for this work was provided by the Spanish Ministry of Economy and Competitiveness (Grants BFU2011-23645 and BFU2014-52656, to M.S. and Doctoral Fellowship BES-2012-052228, to M.B.-O.) and the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa.

PMCID: PMC1276709, 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., This investigation was supported by grants 2RO1 GM27242-24 from the National Institutes of Health and BMC2002-03818 from the Spanish Ministry of Science and Technology to MS and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular 'Severo Ochoa'. VC is holder of a predoctoral fellowship from the Spanish Ministry of Science and Technology, which also supported WJJM by means of the 'Ramón y Cajal' program.

The Bacillus subtilis phage phi29-encoded membrane protein p16.7 is one of the few proteins involved in prokaryotic membrane-associated DNA replication that has been characterized at a functional and biochemical level. In this work we have determined both the solution and crystal structures of its dimeric functional domain, p16.7C. Although the secondary structure of p16.7C is remarkably similar to that of the DNA binding homeodomain, present in proteins belonging to a large family of eukaryotic transcription factors, the tertiary structures of p16.7C and homeodomains are fundamentally different. In fact, p16.7C defines a novel dimeric six-helical fold. We also show that p16.7C can form multimers in solution and that this feature is a key factor for efficient DNA binding. Moreover, a combination of NMR and x-ray approaches, combined with functional analyses of mutants, revealed that multimerization of p16.7C dimers is mediated by a large protein surface that is characterized by a striking self-complementarity. Finally, the structural analyses of the p16.7C dimer and oligomers provide important clues about how protein multimerization and DNA binding are coupled.

Background: The initiation of replication in φ29 mainly occurs opposite the 3' penultimate nucleotide of the template. Results: Mutations in the TP residue Phe-230 changed the specificity for the templating nucleotide. Conclusion: The results suggest a role for Phe-230 in determining the templating nucleotide. Significance: The results shed light on the positioning of the template nucleotide in organisms that initiate with proteinpriming mechanism., Spanish Ministry of Science and Innovation Grant CSD2007–00015 (to M. S.), and a Institutional grant from the Fundación Ramón Areces of the Centro de Biología Molecular

Resolution of the crystallographic structure of φ29 DNA polymerase binary and ternary complexes showed that residue Lys529, located at the C-terminus of the palm subdomain, establishes contacts with the 3′ terminal phosphodiester bond. In this paper, site-directed mutants at this Lys residue were used to analyse its functional importance for the synthetic activities of φ29 DNA polymerase, an enzyme that starts linear φ29 DNA replication using a terminal protein (TP) as primer. Our results show that single replacement of φ29 DNA polymerase residue Lys529 by Ala or Glu decreases the stabilisation of the primer-terminus at the polymerisation active site, impairing both the insertion of the incoming nucleotide when DNA and TP are used as primers and the translocation step required for the next incoming nucleotide incorporation. In addition, combination of the DNA polymerase mutants with a TP derivative at residue Glu233, neighbour to the priming residue Ser232, leads us to infer a direct contact between Lys529 and Glu233 for initiation of TP-DNA replication. Altogether, the results are compatible with a sequential binding of φ29 DNA polymerase residue Lys529 with TP and DNA during replication of TP-DNA. © 2013 del Prado et al., Spanish Ministry of Economy and Competitiveness [grant BFU2011-23645 to M.S.], the Spanish Ministry of Science and Innovation [grant Consolider-Ingenio CSD2007-00015 to M.S.], the Autonomous Community of Madrid [grant S2009MAT-1507 to M.S.], and by an institutional grant from Fundación Ramón Areces to the Centro de Biologı´a Molecular ‘‘Severo Ochoa’’.

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.

Bacteriophage φ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232. The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase. Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives. The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of φ29 TP-DNA replication. These results allow us to propose a role for these residues in the maintenance of the equilibrium between TP-priming domain stabilization and its gradual exit from the polymerization active site of the DNA polymerase as new DNA is being synthesized. © 2012 The Author(s)., Spanish Ministry of Science and Innovation (grants BFU 2008-00215, and Consolider-Ingenio CSD2007-00015).; Autonomous Community of Madrid (grant P-MAT-0283-0505); Fundación Ramón Areces

Uracil-DNA glycosylase (UDG) is a conserved DNA repair enzyme involved in uracil excision from DNA. Here, we report the biochemical characterization of UDG encoded by Bacillus subtilis, a model low G+C Gram-positive organism. The purified enzyme removes uracil preferentially from single-stranded DNA over double-stranded DNA, exhibiting higher preference for U:G than U:A mismatches. Furthermore, we have identified key amino acids necessary for B. subtilis UDG activity. Our results showed that Asp-65 and His-187 are catalytic residues involved in glycosidic bond cleavage, whereas Phe-78 would participate in DNA recognition. Recently, it has been reported that B. subtilis phage φ29 encodes an inhibitor of the UDG enzyme, named protein p56, whose role has been proposed to ensure an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracils present in the φ29 genome. In this work, we also show that a φ29-related phage, GA-1, encodes a p56-like protein with UDG inhibition activity. In addition, mutagenesis analysis revealed that residue Phe-191 of B. subtilis UDG is critical for the interaction with φ29 and GA-1 p56 proteins, suggesting that both proteins have similar mechanism of inhibition.

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., We are grateful to Richard P. Cunningham (Albany, NY) who kindly provided E. coli strain RPC501, and to José M. Lázaro for advice in protein purification. This work was supported by the Spanish Ministry of Science and Innovation Grants Consolider-Ingenio CSD2007-00015 and BFU2008-00215 (to M.S.), by Spanish Research Council Grant 200920I012 (to M.d.V.), and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa.” B.B. is a recipient of a fellowship from Fundación Ramón Areces.

Bacteriophages φ29 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 φ29 and Nf proteins, we show that this domain is the main structural determinant that dictates the internal 3′ nucleotide used as template during initiation., This work was supported by Spanish Ministry of Education and Science Grant BFU 2005-00733 and Autonomous Community of Madrid (Grant P-MAT-0283-0505 (to M.S.) and by an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa.” E.L. was Predoctoral Fellow of the Spanish Ministry of Education and Science

Bacillus subtilis gene yshC encodes a 64 kDa family X DNA polymerase (PolXBs), which contains all the critical residues involved in DNA and nucleotide binding, as well as those responsible for catalysis of DNA polymerization, conserved in most family X members. Biochemical analyses of the purified enzyme indicate that PolXBs is a monomeric and strictly template-directed DNA polymerase, preferentially acting on DNA structures containing gaps from one to few nucleotides and bearing a phosphate group at the 5´ end of the downstream DNA. The fact that PolXBs is able to conduct filling of a single nucleotide gap, allowing further sealing of the resulting nick by a DNA ligase points to a putative role in base excision repair during the B. subtilis life cycle., This work has been aided by the Spanish Ministry of Education and Science grants BFU 2005-00733 and Consolider-Ingenio 2010 24717, and by an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. B.B. is a recipient of a fellowship from Fundación Ramón Areces.

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, P.P-A. and E.L. were predoctoral fellows of the Spanish Ministry of Education and Science. Spanish Ministry of Education and Science (BFU 2005-00733 to M.S.); Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. Funding to pay the Open Access publication charges for this article are provided by research grant BFU 2005-00733 from the Spanish Ministry of Education and Science

Remarkably little is known about the in vivo organization of membrane-associated prokaryotic DNA replication or the proteins involved. We have studied this fundamental process using the Bacillus subtilis phage 29 as a model system. Previously, we demonstrated that the 29-encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane-associated 29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the 29 terminal protein. In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C-terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein. Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA-binding mode. These and previous results indicate that p16.7 encompasses four distinct modules. An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo 29 DNA replication is presented., This investigation was supported by National Institutes of Health Research Grant 2RO1 GM27242-23, Dirección General de Investigación Científica y Técnica Grant PB98-0645 and an Institutional grant from Fundación Ramón Areces to the Centro Biología Molecular ‘Severo Ochoa’. A.S.R and D.M. were holders of a predoctoral fellowship from ‘Gobierno Vasco’ and ‘Fondo de Investigaciones Sanitarias’, respectively. The ‘Ramón y Cajal’ program of the Spanish ministry of Science and Technology supported W.J.J.M.

Prokaryotic DNA replication is compartmentalized at the cellular membrane. The Bacillus subtilis phage varphi29-encoded membrane protein p16.7 is one of the few proteins known to be involved in the organization of prokaryotic membrane-associated DNA replication. The functional DNA binding domain of p16.7 is constituted by its C-terminal half, p16.7C, which forms high affinity dimers in solution and which can form higher order oligomers. Recently, the solution and crystal structures of p16.7C and the crystal structure of the p16.7C-DNA complex have been solved. Here, we have studied the p16.7C dimerization process and the structural and functional roles of p16.7 residues Trp-116 and Asn-120 and its last nine C-terminal amino acids, which form an extended tail. The results obtained show that transition of folded dimers into unfolded monomers occurs without stable intermediates and that both Trp-116 and the C-terminal tail are important for dimerization and functionality of p16.7C. Residue Trp-116 is involved in formation of a novel aromatic cage dimerization motif, which we call "Pro cage." Finally, whereas residue Asn-120 plays a minor role in p16.7C dimerization, we show that it is critical for both oligomerization and DNA binding, providing further evidence that DNA binding and oligomerization of p16.7C are coupled processes.

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., This investigation was supported by grants BFU2005-00733 and BFU2005-01878 from the Spanish Ministry of Education and Science to MS and WJJM, respectively, and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular 'Severo Ochoa'. VC and DM are holders of predoctoral fellowships from the Spanish Ministry of Science and Technology and the Fondo de Investigación Sanitaria, respectively. WJJM was supported by means of the 'Ramón y Cajal' program from the Spanish Ministry of Science and Technology.

The Bacillus subtilis phage phi29-encoded membrane protein p16.7 is one of the few proteins involved in prokaryotic membrane-associated DNA replication that has been characterized at a functional and biochemical level. In this work we have determined both the solution and crystal structures of its dimeric functional domain, p16.7C. Although the secondary structure of p16.7C is remarkably similar to that of the DNA binding homeodomain, present in proteins belonging to a large family of eukaryotic transcription factors, the tertiary structures of p16.7C and homeodomains are fundamentally different. In fact, p16.7C defines a novel dimeric six-helical fold. We also show that p16.7C can form multimers in solution and that this feature is a key factor for efficient DNA binding. Moreover, a combination of NMR and x-ray approaches, combined with functional analyses of mutants, revealed that multimerization of p16.7C dimers is mediated by a large protein surface that is characterized by a striking self-complementarity. Finally, the structural analyses of the p16.7C dimer and oligomers provide important clues about how protein multimerization and DNA binding are coupled.

The Bacillus subtilis phage φ29-encoded membrane protein p16.7 is one of the few proteins known to be involved in prokaryotic membrane-associated DNA replication. Protein p16.7 contains an N-terminal transmembrane domain responsible for membrane localization. A soluble variant lacking the N-terminal membrane anchor, p16.7A, forms dimers in solution, binds to DNA, and has affinity for the φ29 terminal protein. Here we show that the soluble N-terminal half of p16.7A can form a dimeric coiled coil. However, a second domain, located in the C-terminal half of the protein, has been characterized as being the main domain responsible for p16.7 dimerization. This 70-residue C-terminal domain, named p16.7C, also constitutes the functional part of the protein as it binds to DNA and terminal protein. Sequence alignments, secondary structure predictions, and spectroscopic analyses suggest that p16.7C is evolutionarily related to DNA binding homeodomains, present in many eukaryotic transcriptional regulator proteins. Based on the results, a structural model of p16.7 is presented., This work was supported in part by Research Grant 2RO1 GM27242-24 from the National Institutes of Health (to M. S.) and by Grants BIO2003-04445 (to M. G. M.) and BMC2002-03818 (to M. S.) from the Spanish Ministry of Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages φ29 and Nf. In this work, the self-interaction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo cross-linking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of φ29 and Nf. Three deletion mutant proteins have been characterized, ΔN19, ΔN26, and ΔN33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein ΔN19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins ΔN26 and ΔN33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein., This work was supported by Grant 2R01 GM27242-23 from the National Institutes of Health, Grants PB-98-0645 and PB96-818 from Dirección General de Investigación Cientı́fica y Técnica, and an institutional grant from Fundación Ramón Areces to the Centro de Biologı́a Molecular “Severo Ochoa.”The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A new self-sustained terminal protein-primed DNA amplification system has been used to describe in vitro evolutionary changes affecting maintenance of the genome size of bacteriophage φ29. These changes involve generation and efficient amplification of short palindromic molecules containing an inverted duplication of one of the original DNA ends. A template-switching mechanism is proposed to account for the appearance of these molecules. After their formation, they would replicate by means of hairpin intermediates. Relevant kinetic information about this DNA replication system has been obtained from the competition between the input full-length φ29 DNA and its derived truncated versions. The physiological relevance of these molecules and the mechanisms to control their formation are discussed., This investigation has been aided by Research Grant 5R01 GM27242-17 from the National Institutes of Health, by Grant PB93- 0173 from the Direccio´n General de Investigacio´n Cientı´fica y Te´cnica, by Grant CHRX-CT 93-0248 from the European Economic Community, and by an Institutional grant from Fundacio´n Ramo´n Areces. J.M. was recipient of a postdoctoral fellowship from the Comunidad Auto´noma de Madrid.

Summary Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance., Graphical Abstract, Highlights • Primer-independent PolBs (piPolBs) display templated de novo DNA synthesis capacity • piPolBs denote a third major group of family B DNA polymerases • piPolBs are the hallmark of pipolins, self-replicating mobile genetic elements • Pipolins are widespread among diverse bacterial phyla and mitochondria, Redrejo-Rodríguez et al. report and characterize a DNA polymerase group (piPolB) from the B family that can perform primer-independent DNA replication. PiPolBs are encoded by Pipolins, diverse self-replicating genetic elements that are widespread among bacterial phyla and in mitochondria.