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 phi29 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 phi29 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 phi29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses phi29 development by repressing the main early phi29 promoters via different and novel mechanisms and also by preventing activation of the single late phi29 promoter.

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.

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...

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To initiate ϕ29 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 ϕ29 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 ϕ29 DNA polymerase containing the GA-1 TPR-1 subdomain could use GA-1 TP, but only in the presence of ϕ29 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 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 bacterium Bacillus subtilis (PolX Bs ) 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 PolX Bs 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. apurinic/apyrimidinic-lyase | site-directed mutagenesis G enomes are continuously insulted by exogenous and endogenous genotoxic agents, as ionizing radiation, drugs, and (by)products of normal cellular metabolism that generate reactive oxygen species (ROS) leading to mainly nonbulky DNA lesions (1). Base excision repair (BER) is the major pathway involved in the removal of this type of damage, and its importance for cell survival is reflected by its conservation from bacteria to eukaryotes (2). During the first steps of BER, highly mutagenic apurinic/apyrimidinic (AP) intermediates are produced as a result of hydrolytic cleavage of the altered base-sugar bond by mono-(class II) and/or bifunctional (class I) DNA N-glycosylases (ref. 3 and references therein), or from spontaneous DNA base loss, causing replication and transcription inhibition if left unrepaired (4, 5). AP endonucleases play a crucial role in BER because they recognize the abasic residue and hydrolyze the phosphodiester bond 5′ to the AP site, leaving a gapped DNA intermediate with an extendable 3′-OH end (ref. 2 and references therein).Members of the family X of DNA polymerases (hereafter, PolX) are widely spread in nature from virus to humans, being involved in the DNA synthesis step during BER and DNA double-strand break repair by virtue of a common Polβ-like core adapted to fill the gapped DNA intermediates very proficiently (6-9).PolX Bs (570-aa long) is a prototypic bacterial/archaeal PolX member from Bacillus subtilis with a N-terminal Polβ-like core (residues 1-317) responsible for catalysis of DNA polymerization (10), and a C-terminal polymerase and histidinol phosphatase (PHP) domain (residues 333-570) containing highly conserved residues that catalyze a Mn 2þ -dependent 3′-5′-exonuclease activity (11-14), which shows a preferential processing of unannealed 3′ termini (12). Due to this fact and to its adaptation to perform filling of small gaps (10), PolX Bs was proposed to play a potential role in the DNA synthesis step of repair p...

A.Serna-Rico and D.Mun Äoz-Espõ Ân contributed equally to this workRemarkably 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 f29 as a model system. Previously, we demonstrated that the f29-encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane-associated f29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the f29 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 f29 DNA replication is presented. Keywords: Bacillus subtilis/bacteriophage f29/in vivo DNA replication/ssDNA binding/terminal protein interaction IntroductionEukaryotic DNA replication occurs at numerous ®xed positions within the nucleus, as assessed by microscopic imaging techniques, implying that they are attached to subcellular structures (reviewed in Cook, 1999). During recent years, microscopic imaging tools have been developed for prokaryotic research and the results obtained have contributed importantly to a better understanding of in vivo prokaryotic DNA replication and related processes (Jensen and Shapiro, 2000). One of the most important recent contributions is the discovery that replicative DNA polymerase of Bacillus subtilis is located at relatively stationary cellular positions (Lemon and Grossman, 1998). This study had a vast impact on the view of prokaryotic DNA replication. First, it implied that the replicating DNA template moves through the stationary polymerase, contrary to the generally accepted view that the DNA polymerase moves along the DNA during replication. Second, it indicated that DNA polymerase, together with other proteins involved in DNA replication, are organized in so-called stationary replication factories. Finally, the stationary position of the replication factory entails that it is attached to a substructure. This adapted view of DNA replication, which most probably applies to all bacteria, shows remarkable similarities to that of eukaryotic DNA replication (reviewed in Cook, 1999), indicating that the basic principles of prokaryotic and eukaryotic DNA replication are more conserved than was previously thought.Compelling evidence has been provided during the past few decades that prokaryotic DNA replication, including that of resident plasmids and infecting phages, occurs at the cellular membrane (for review see Firshein, 1989), which most probably is the ...