DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5′ ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3′–5′ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the TP at the bacterial nucleoid, where viral DNA replication takes place. The biochemical properties of the Φ29 DBP and SSB and their function in the initiation and elongation of Φ29 DNA replication, respectively, will be described.


INTRODUCTION
Bacteriophages are the most abundant biological entities on earth (Brüssow and Hendrix, 2002). Approximately 96% of the reported bacteriophages belong to the order Caudovirales, which is composed of three families: Myoviridae, Siphoviridae, and Podoviridae (Ackermann, 2003). Bacillus subtilis phage 29 belongs to the Podoviridae family and to the Φ29-like genus, together with phages 15, PZA, BS32, B103, Nf, M2Y, and GA-1 (Ackermann, 1998). These are the smallest phages that infect Bacillus, and they are among the smallest known phages that possess a dsDNA genome (Anderson and Reilly, 1993). Based on its relatedness, these phages have been classified in three groups: group I includes phages 29, PZA, 15, and BS32; group II contains phages B103, Nf and M2Y; and group III has GA-1 as its only member (Yoshikawa et al., 1985(Yoshikawa et al., , 1986Pecenkova and Paces, 1999).
Bacteriophage 29 genome consists of a linear dsDNA ∼19 Kb-long with a terminal protein (TP) covalently linked to each 5 ′ end (Salas, 1991). 29 has served as a model system for studying the protein-priming mechanism of DNA replication, being the TP-primed replication system best characterized in vitro. The use of a TP as primer for viral DNA replication has also been described for other bacteriophages (e.g., Escherichia coli and Streptococcus pneumoniae phages PRD1 and Cp-1, respectively), eukaryotic viruses (adenovirus), and some Streptomyces spp. (Chang and Cohen, 1994;Bao and Cohen, 2001). In addition, the presence of TPs has been described or suggested in viruses infecting Archaea (Bath et al., 2006;Peng et al., 2007), some linear plasmids of bacteria, fungi, and higher plants (Salas, 1991;Meinhardt et al., 1997;Chaconas and Chen, 2005), transposable elements (Kapitonov and Jurka, 2006) and mitochondrial DNA (Fricova et al., 2010).
Besides the essential role of priming DNA replication, TPs can perform additional functions. It has been shown that adenovirus TP is important for the anchoring of the viral genome to the nuclear matrix, which enhances transcription of the viral DNA (Schaack et al., 1990). TPs have also been shown to be required for DNA packaging (Bjornsti et al., 1982(Bjornsti et al., , 1983, transfection (Hirokawa, 1972;Ronda et al., 1983;Porter and Dyall-Smith, 2008), and nucleoid and nuclear targeting (Tsai et al., 2008;Muñoz-Espín et al., 2010;Redrejo-Rodríguez et al., 2012). Furthermore, biochemical studies have suggested that 29 TP is endowed with peptidoglycan-hydrolytic activity (Moak and Molineux, 2004).

TERMINAL PROTEIN
Replication of the 29 genome takes place by a process of symmetrical replication in which both origins are used for initiation in a non-simultaneous manner ; Figure 1). The protein that acts as primer for the initiation of 29 DNA replication, the so-called TP, is a 266 amino acids protein encoded by the early viral gene 3. The first step of 29 DNA replication is the formation of a heterodimer between a free molecule of TP (primer TP) and the DNA polymerase ( 29 DNAP) . Then, this complex recognizes the replication origins, located at both ends of the viral genome, by specific interactions with both the TP that is linked to the genome ends by a previous round of replication (parental TP) and DNA sequences (García et al., 1984;Gutiérrez et al., 1986a,b;González-Huici et al., 2000a,b). The parental TP is the major signal for replication origin recognition by the heterodimer (Gutiérrez et al., 1986b;González-Huici et al., 2000b) and both, DNA polymerase and primer TP, are involved in such recognition through specific interactions with the parental TP (Freire et al., 1996;Illana et al., 1998;González-Huici et al., 2000a;Serna-Rico et al., 2000;Pérez-Arnáiz et al., 2007). The 29 double-stranded DNA binding protein p6 (DBP) (see below) binds all along 29 DNA forming a nucleoprotein complex that causes the unwinding of the DNA helix at the ends, facilitating the initiation step (Serrano et al., 1994). After origin recognition, the viral DNA polymerase catalyzes the formation of a phosphoester between the first dAMP and the hydroxyl group of the primer TP residue Ser232 Hermoso et al., 1985). The initiation reaction is directed by the second T at the 3 ′ end of the template (3 ′ TTTCAT 5 ′ ), after which the TP-dAMP complex translocates one position backwards to recover the information corresponding to the first T of the template strand. Then, the second T will serve again as template for the incorporation of the following nucleotide (Méndez et al., 1992). This backward translocation of the TP-dAMP complex is known as sliding-back mechanism and requires a terminal repetition of at least 2 nucleotides in the template strand to guarantee the fidelity of the initiation reaction (Méndez et al., 1992) (see below). The TP/DNA polymerase heterodimer is not dissociated immediately after initiation. There is a transition stage in which the DNA polymerase synthesizes a 5 nt-long elongation product while complexed with TP, undergoes some structural changes during the incorporation of nucleotides 6 to 9, and finally dissociates from the TP after the incorporation of the 10th nucleotide (Méndez et al., 1997). Then, the viral DNA polymerase continues DNA elongation in a processive manner, which occurs coupled to the displacement of the nontemplate strand . DNA elongation by one 29 DNAP coming from each origin generates type I replicative intermediates, consisting of full-length 29 dsDNA molecules with two branches of ssDNA. These stretches of ssDNA are bound by the viral single-stranded DNA-binding protein p5 (SSB) (see below) (Gutiérrez et al., 1991), which will be further removed during the polymerization process. When the two replication forks meet, the type I replicative intermediate gives rise to two physically separated type II replicative intermediates. These molecules consist of full-length 29 DNA in which a portion of the DNA starting from one end is dsDNA and the portion spanning to the other end is ssDNA (Harding and Ito, 1980;Inciarte et al., 1980). Termination of viral DNA replication occurs when the DNA polymerase reaches the template end, and after replication of the last nucleotide, dissociates from the viral genome.
The 3.0 Å resolution crystallographic structure of the heterodimer formed between 29 DNA polymerase and TP revealed that the latter is composed of three structural domains (Kamtekar et al., 2006; see Figure 2): • The TP N-terminal domain comprises residues 1 to 73 and its tertiary structure is unknown because it was disordered in the crystal lattice. Circular dichroism experiments have shown that this domain has a high content in αhelix (60%), and secondary structure predictions determined two αhelices connected by a disordered loop (Holguera et al., 2014). This FIGURE 1 | Schematic representation of the bacteriophage 29 DNA replication mechanism. 29 DNA replication starts non-simultaneously at both DNA ends. The TP/DNA polymerase heterodimer recognizes the p6-complexed replication origins and the DNA polymerase catalyzes the covalent linkage of dAMP to TP residue Ser232 (initiation reaction). After a transition step (not drawn in the figure), the DNA polymerase dissociates from the TP and continues processive elongation coupled to strand displacement. Viral protein p5 binds to the displaced ssDNA strands and is further removed during the polymerization process. Continuous elongation by two DNA polymerases gives rise to the complete duplication of the parental strands. Green ovals: parental TP; black ovals: primer TP; red circles: p6; blue: DNA polymerase; yellow ovals: SSB p5. Linear dsDNA is shown as a double helix. Adapted from de Vega and  domain is responsible for non-sequence specific DNA binding (Zaballos and Salas, 1989) and for the localization of the protein at the bacterial nucleoid (Muñoz-Espín et al., 2010). In addition, a role in origin unwinding has been proposed for the TP N-terminal domain, since this domain is not required for the initiation reaction at a partially open origin (Pérez-Arnáiz et al., 2007;Gella et al., 2014).  (Kamtekar et al., 2006;del Prado et al., 2012) and is responsible for dictating the nucleotide used as template during initiation of viral DNA replication (Longás et al., 2008). The TP priming domain has been proposed to mimic duplex product DNA in its electrostatic profile and binding site in the DNA polymerase, as both occupy the same binding cleft in the DNA polymerase (de Vega et al., 1998a;Kamtekar et al., 2006).
There are not proteins in structural databases with sufficient structural homology with 29 TP. Genes encoding TPs from other Φ29-like phages such as B103, PZA, Nf, and GA-1 have been sequenced (Paces et al., 1985;Leavitt and Ito, 1987;Illana et al., 1996;Pecenková et al., 1997;Meijer et al., 2001). The amino acid sequence comparison of these TPs has revealed a high degree of conservation between PZA and 29 TPs (97.7% identity). In fact, 29 TP can functionally substitute for PZA TP in vivo (Bravo et al., 1994a). The conservation is lower in the case of Nf (62.4% identity) and B103 (62% identity) TPs (Leavitt and Ito, 1987;Pecenková et al., 1997). GA-1 TP is the most distantly related one, being the percentage of identity with 29 TP of 40% (Illana et al., 1996).

TP Residues Involved in Priming Activity
Site-directed mutagenesis has been carried out at the TP priming residue Ser232. The change of Ser232 to Thr gives rise to a protein completely inactive in the initiation reaction (Garmendia et al., 1988). Similarly, the change of Ser232 into Cys almost completely abolishes the initiation capacity of the TP mutant, being its initiation capacity about 1% of that of the wild-type TP (Garmendia et al., 1990). These TP mutants interacted in a wildtype manner with both 29 DNAP and DNA (Garmendia et al., 1988(Garmendia et al., , 1990. Furthermore, mutation of TP priming-loop residues Leu220 and Ser226 into Pro highly impaired the initiation activity but did not affect either DNA polymerase or DNA-binding, suggesting the implication of these residues in the initiation reaction (Garmendia et al., 1990).

TP Residues Involved in DNA-Binding
29 TP binds to both single-stranded and double-stranded DNA in vitro (Prieto, 1986;Zaballos and Salas, 1989). As mentioned above, the TP domain responsible for non-specific DNA-binding is the N-terminal domain (Zaballos and Salas, 1989). As in many non-sequence specific DNA-binding proteins, TP N-terminal domain basic residues are implicated in its DNA-binding capacity (Holguera et al., 2014). Viral DNA replication in prokaryotes takes place at specific subcellular locations. In this sense, the use of host organizing structures seems to be essential to provide an appropriate scaffold for viral DNA replication. 29 TP localizes at the bacterial nucleoid along the infective cycle, being the N-terminal domain responsible for this localization (Muñoz-Espín et al., 2010). Additionally, parental TP (and therefore TP-DNA) localizes at the bacterial nucleoid, independently of primer TP (Muñoz-Espín et al., 2010). Importantly, the TP N-terminal domain is essential for an efficient viral DNA replication in vivo (Muñoz-Espín et al., 2010). To determine the TP residues involved in nucleoid targeting, each basic residue of the TP Nterminal domain was replaced independently by alanine, and the subcellular localization of the resulting proteins fused to YFP was analyzed. Lys27 was the only TP residue that, changed individually, impaired the TP nucleoid localization (Holguera et al., 2014). By using X-Chip techniques, it was shown that wild-type 29 TP, but not mutant K27A, binds B. subtilis genome in vivo, establishing a correlation between nucleoid localization and DNA-binding (Holguera et al., 2014). During the infective cycle both TP and viral DNA polymerase localize at the bacterial nucleoid, being the nucleoid localization of the DNA polymerase dependent on the expression of TP (Muñoz-Espín et al., 2010). The subcellular localization of the viral DNA replication machinery at the bacterial nucleoid has been proposed to serve as a compartmentalization mechanism to make the replication process more efficient, as well as a means of taking advantage of the bacterial chromosome segregation dynamics (Muñoz-Espín et al., 2010). The impact of bacterial chromosome TP binding on host processes such as DNA replication and transcription remains to be investigated.
Interestingly, 29 TP localizes at the bacterial nucleoid when expressed in the distantly related bacterium E. coli, being the TP N-terminal domain the one responsible for this localization (Muñoz-Espín et al., 2010;Redrejo-Rodríguez et al., 2013). Furthermore, the TP from phage PRD1, which infects E. coli among other bacteria, localizes at the E. coli nucleoid independently of other viral components. TPs from other phages such as Cp-1, Nf, and GA-1 also localize at the E. coli nucleoid, although localization in their host systems remains to be determined (Redrejo- Rodríguez et al., 2013). Altogether, these results suggest that nucleoid localization is a functional property conserved in phage TPs. Importantly, a Nuclear Localization Signal (NLS) has been described in 29 TP, as well as in a variety of other TPs such as those from Nf, PRD1, Bam35, and Cp-1 phages (Redrejo- Rodríguez et al., 2012).

TP Residues Involved in DNA Polymerase-Binding
The extensive interactions of 29 TP intermediate and priming domains with the DNA polymerase account for the high stability of the heterodimer (Lázaro et al., 1995;Kamtekar et al., 2006). The crystallographic structure of the heterodimer shows that the TP intermediate domain is structurally complementary to the DNA polymerase TPR1 subdomain; this interface has many charged residues that include two salt bridges between TP residues Arg158 and Arg169, and DNA polymerase residues Glu291 and Glu322, respectively (Kamtekar et al., 2006; Figure 2A). In the case of the highly electronegative TP priming domain, the structure shows interactions between TP residues Glu191 and Asp198, and DNA polymerase thumb subdomain residues Lys575 and Lys557, respectively ( Figure 2B). In addition, TP residues Gln253 and Tyr250 would interact with DNA polymerase exonuclease domain residue Arg96 through a hydrogen bond and a stacking interaction, respectively (Kamtekar et al., 2006; Figure 2C). In this sense, mutation of DNA polymerase residue Arg96 to alanine was shown to impair the interaction with TP (Rodríguez et al., 2004). Similarly, TP residues Glu252, Gln253, and Arg256 from the C-terminal helix of the priming domain would pack against DNA polymerase TPR2 subdomain residues Leu416, Gly417, and Glu419, respectively (Kamtekar et al., 2006;Figures 2C,D). In fact, by biochemical analysis of TP mutants, TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253, and Arg256 were shown to be involved in the interaction between TP and DNA polymerase (del Prado et al., 2012). Additionally, biochemical studies using both TP and DNA polymerase mutant proteins strongly suggest that TP priming loop residue Glu233 interacts directly with the DNA polymerase palm subdomain residue Lys529 during the first step of TP-DNA replication (del Prado et al., 2013).

TP Interaction with Other Viral Proteins
Apart from the DNA polymerase, 29 TP interacts with other viral proteins. By means of in vitro chemical crosslinking, it has been shown that 29 TP interacts with the viral early protein p1, which is a membrane-associated protein. Based on these results, a model of membrane anchorage of the viral replication machinery mediated by p1 has been proposed (Bravo et al., 2000). In addition, TP interacts with the membrane protein p16.7 in vitro (Serna-Rico et al., 2003), presenting another anchoring point to the bacterial membrane. This interaction has also been proposed to facilitate the binding of p16.7 to the displaced strands of the viral genome, favoring their recruitment to the bacterial membrane (Serna-Rico et al., 2003). Mutations introduced at several residues of the TP N-terminal and intermediate domains impaired DNA replication when TP acted simultaneously as primer and parental TP, suggesting that a proper interaction between primer and parental TP is important for origin recognition (Illana et al., 1999;Serna-Rico et al., 2000;del Prado et al., 2012;Holguera et al., 2015).

DNA POLYMERASE
Processive Polymerization Coupled to Strand Displacement: Two Specific Attributes of 29 DNA Polymerase 29 DNAP is a small (66 kDa) single subunit enzyme, the product of the viral gene 2, characterized as the viral DNA replicase Salas, 1984, 1985b;Salas, 1991), and belonging to the family B (eukaryotic-type) of DNA-dependent DNA polymerases Bernad et al., 1987). As any other conventional DNA polymerase, 29 DNAP catalyzes the sequential template-directed addition of dNMP units onto the 3 ′ -OH group of a growing DNA chain in a faithful manner as it shows discrimination values of 10 4 -10 6 , and a poor mismatch elongation efficiency (Esteban et al., 1993). Extensive site directed mutagenesis studies in 29 DNAP described the function of specific amino acids at motifs YxGG, Dx 2 SLYP, LExE, Kx 3 NSxYG, Tx 2 GR, YxDTDS, and KxY, placed at the C-terminal domain (residues 190-572; polymerization domain) and highly conserved among the eukaryotic DNA polymerases from family B Salas, 1995, 1996;Pérez-Arnaiz et al., 2006Salas and de Vega, 2006;del Prado et al., 2013;Santos et al., 2014). These investigations allowed the identification of the catalytic residues responsible for coordinating the metal ions and the ones acting as ligands of the substrates (DNA, TP, and dNTP).
In contrast to the complexity of other in vitro replication systems, efficient synthesis of full-length 29 TP-DNA can be accomplished in vitro with only the presence of TP and 29 DNAP (Blanco and Salas, 1985b). The efficiency of this minimal replication system relies on three unique catalytic features of 29 DNAP: (1) ability to initiate DNA replication by using a TP as primer (Salas, 1991), thus bypassing the need for a primase (see below). (2) an extremely high processivity (>70 kb, measured by rolling circle replication, the highest described for a DNA polymerase), allowing replication of the entire genome from a single binding (and priming) event, without the assistance of processivity factors ; (3) unlike most replicases, 29 DNAP efficiently couples DNA polymerization to strand displacement, without the need of helicase-like proteins . These three aforementioned exceptional properties are essential to allow the symmetric DNA replication mode of bacteriophage 29 mentioned above, by which the two DNA strands are synthesized continuously from both ends of the linear molecule . In the case of 29 TP-DNA amplification, the single-stranded DNA binding protein p5 and the double-stranded DNA binding protein p6 are essential.
Resolution of the 29 DNAP structure, in collaboration with Thomas Seitz's lab (Yale University), gave the insights into these three unique properties of the enzyme, use of TP as primer, processivity, and strand displacement capacity (Kamtekar et al., 2004(Kamtekar et al., , 2006. Thus, the 29 DNAP structure is formed by an N-terminal exonuclease domain, harboring the 3 ′ -5 ′ exonuclease active site, and a C-terminal polymerization domain (see Figure 3A) that has the universally conserved palm (containing the catalytic residues as well as DNA ligands), fingers (mainly involved in binding the incoming dNTP), and thumb (containing DNA ligands which confer stability to the primer-terminus) subdomains (Kamtekar et al., 2004). Although a priori this bimodular structure would be a common theme among proofreading DNA polymerases, the main structural novelty was the presence in the polymerization domain of 29 DNAP of two subdomains called TPR1 and TPR2, specifically present in the protein-primed subgroup of DNA polymerases (Blasco et al., 1990;Dufour et al., 2000). TPR1 is placed at the edge of the palm, while TPR2 contains a β-hairpin and forms with the apex of the thumb subdomain an arch-like structure. Palm, thumb, TPR1, and TPR2 subdomains form doughnutshaped structure at the polymerization active site that encircles the growing DNA product (Berman et al., 2007), acting as an internal clamp that confers the DNA-binding stability responsible for the inherent processivity of the enzyme (Rodríguez et al., 2005), similar to the sliding clamps used by other replicative polymerases (see Figure 3B). On the other hand, TPR2, palm and fingers subdomains, together with the exonuclease domain, encircle the downstream template strand (Berman et al., 2007), forming a narrow tunnel whose dimensions (∼10 Å) do not allow dsDNA binding. This fact forces the unwinding of the downstream dsDNA to allow threading of the template strand through this tunnel to reach the polymerization site, using the same topological mechanism as hexameric helicases to open dsDNA regions (see Figure 3B), providing the structural basis for the strand displacement capacity of 29 DNAP (Kamtekar et al., 2004;Rodríguez et al., 2005). The use of optical tweezers has allowed to conclude that the DNA polymerase destabilizes the two nearest base pairs of the fork by maintaining a sharp bending of the template and the complementary strands at a closed fork junction (Morin et al., 2012). Therefore, the polymerase, instead of behaving as a "passive" unwinding motor that would imply that translocation of the protein traps transient unwinding fluctuations of the fork, behaves as an FIGURE 3 | (A) Ribbon Representation of the Domain Organization of 29 DNAP. The exonuclease domain is shown in red, the palm in pink, TPR1 in gold, the fingers in blue, TPR2 in cyan, and the thumb in green. Asp249 and Asp458, which provide the catalytic carboxylates of the polymerase active site, are shown using space-filling spheres. Reproduced with permission from Kamtekar et al. (2004). (B) Modeling processivity and strand displacement in 29 DNAP. The TPR2 insertion would contribute to a full encirclement of the DNA substrate, conferring a remarkable processivity, and also acts as a structural barrier, which would force the DNA strands of the parental DNA to diverge (melt). Because 29 DNAP translocates after each polymerization cycle, the TPR2 subdomain would act as a wedge to couple polymerization to strand displacement. Reproduced with permission from Rodríguez et al. (2005). Copyright (2005) National Academy of Sciences, U.S.A.
"active" motor, actively destabilizing the duplex DNA at the junction.

On the Translocation Mechanism of 29 DNA Polymerase
As any other replicative DNA polymerase, after inserting a dNMP, 29 DNAP has to translocate the growing DNA one position backwards to allow the next insertion step to occur, a process called translocation. The structures of the binary and ternary complexes of 29 DNAP provided a structural basis for comprehending the mechanism of translocation (Berman et al., 2007). The dNTP insertion site is initially occupied by the aromatic ring of two conserved residues, Tyr390 (from the fingers subdomain) and Tyr254 (from the palm subdomain; see Figure 4). Once the incoming nucleotide gains access and binds at the polymerization active site it triggers a 14 • rotation of the fingers subdomain toward the polymerization active site, going from an open to a closed state and allowing electropositively charged residues from the fingers subdomain to bind the α-and β-phosphates of the dNTP. Closing of the fingers provokes Tyr390 and Tyr254 to abandon the nucleotide insertion site to form part of the nascent base pair binding pocket, allowing the base moiety of the incoming nucleotide to form a Watson-Crick base pair with the templating nucleotide, whereas the deoxyribose ring stacks on the phenolic group of Tyr254. Once the phosphoester bond formation between the α-phosphate of the incoming dNTP and the OH-group of the priming nucleotide has taken place (pre-translocation state), the pyrophosphate produced leaves the DNA polymerase, breaking the electrostatic crosslink that kept the fingers subdomain in the closed state. Concomitantly to the fingers opening, residues Tyr254 and Tyr390 move back into the nucleotide insertion site, and the nascent base pair translocates backwards one position (post-translocation state; Berman et al., 2007). This translocation allows the 3 ′ OHgroup of the newly added nucleotide to be in a competent position to prime the following nucleotide insertion event. Direct observation of translocation in individual 29 DNAP complexes monitored with single nucleotide resolution and using the hemolysin nanopore, has allowed to conclude that 29 DNAP translocation occurs discretely from the pre-translocation state to the post-translocation state, driven by Brownian thermal motion (Dahl et al., 2012). Although nucleotide does not drive translocation, the fluctuation of the binary complexes between the pre-translocation and post-translocation states is rectified to the post-translocation state by the binding of complementary dNTP. The movement from the open, post-translocation state, to the closed pre-translocation state most probably reflects an equilibrium between the fingers-open and fingers-closed states to relieve the steric clash of the primer-terminus with residues Tyr254 and Tyr390 (see above), which occlude the nucleotide insertion site when the fingers are open (Dahl et al., 2012). In addition to the synthetic activities described above, φ29 DNAP catalyzes two degradative reactions: 1. Pyrophosphorolysis. 29 DNAP possesses an inorganic pyrophosphate-dependent degradative activity, pyrophosphorolysis (Blasco et al., 1991). This activity, whose optimal substrate is a duplex DNA with a protruding 5 ′ single strand, can be considered as the reversal of polymerization as it acts in the 3 ′ -5 ′ direction releasing free dNTPs by addition of PPi as substrate, in the presence of divalent metal ions, probably playing some role in fidelity (Blasco et al., 1991). The fact that 29 DNAP mutants at the catalytic amino acid residues involved in the DNA polymerization activity were also deficient in the pyrophosphorolytic activity indicated that both activities share a common polymerization active site (Blasco et al., 1991;Santos et al., 2014). 2. 3 ′ -5 ′ exonuclease. A reaction found in the N-terminal domain of the polymerase, and that requires two divalent metal ions to release dNMP units from the 3 ′ end of a DNA strand at a catalytic rate of 500 s −1 . As in other replicases, the 3 ′ -5 ′ exonuclease of 29 DNAP proofreads DNA insertion errors, as it degrades preferentially mismatched primer termini (Blanco and Salas, 1985a;Garmendia et al., 1992).
Sequence alignments and extensive site directed mutagenesis studies carried out during the last three decades in 29 DNAP have been pioneer in the identification and role of the catalytic and ssDNA ligand residues responsible for the 3 ′ -5 ′ exonuclease (reviewed in Salas, 1995, 1996). The presence of homologous residues among distantly related DNA-dependent DNA polymerases allowed us to propose the evolutionary conservation of 3 ′ -5 ′ exonuclease active site  in the proofreading DNA polymerases. Thus, the exonuclease active site, located at the N-terminal domain (residues 1-189; exonuclease domain, see Figure 3), is formed by three conserved N-terminal amino acid motifs, ExoI, ExoII, and ExoIII, that contain four carboxylate groups (Asp12, Glu14, Asp66, and Asp169 in 29 DNAP) that coordinate two metal ions, and one tyrosine residue (Tyr165 in 29 DNAP) that orients the attacking water molecule . Moreover, these analyses allowed the identification of a new motif (Kx 2 hxA), specifically conserved in family B DNA polymerases and whose lysine residue ( 29 DNAP Lys143) plays an auxiliary role in catalysis (de Vega et al., 1997), stabilizing the catalytic Asp169 of the Exo III motif (Berman et al., 2007). Crystallographic resolution of 29 DNAP with a ssDNA at the exonuclease active site demonstrated the existence of two stable conformations at the exonuclease active site of family B DNA polymerases (see Figure 5), as previously suggested from comparisons of T4 and RB69 DNA polymerase exonuclease structures with the E. coli DNA polymerase I Klenow fragment exonuclease structure (Beese and Steitz, 1991;Wang et al., 1996Wang et al., , 2004. In one conformation, the tyrosine from the Exo III motif (Tyr165 in 29 DNAP) is solvent exposed, whereas in the other conformation, it contacts with the scissile phosphate through the nucleophile while conserved lysine from motif Kx 2 hxA ( 29 DNAP Lys143) stabilizes the catalytic aspartate of the Exo III motif ( 29 DNAP Asp169), consistent with the previous biochemical results (de Vega et al., 1997). The latter conformation seems to be the more chemically and biologically relevant complex for exonuclease activity. The two conformations observed suggest that the movement of the conserved tyrosine and lysine residues into the active site sets up the active site for the exonucleolysis reaction in the family B DNA polymerases.
A tight and fine-tuned coordination between the polymerization and exonucleolytic cycles should take place to allow a productive and faithful replication. Previous studies showed that 29 DNAP proofreads the misinserted nucleotides intramolecularly (de Vega et al., 1999). This fact implies that the DNA polymerase transfers the mismatched 3 ′ -teminus to the 3 ′ -5 ′ exonuclease active site for erroneous dNMP release without dissociating from the DNA. Comparison of DNA polymerase structures of RB69 DNA polymerase in polymerization and editing modes showed that the primer-terminus switches between both active sites by the rotation of a top microdomain of the thumb subdomain (Shamoo and Steitz, 1999;Franklin et al., 2001). However, in the 3D resolution of 29 DNAP structure the thumb subdomain has an unusual structure mainly constituted by a static long β-hairpin that does not rotate upon DNA binding (Kamtekar et al., 2004;Berman et al., 2007, see Figure 3). In addition, the blockage of the thumb movements by introducing a disulfide bond between the tips of the TPR2 and thumb subdomains had not effect in the partitioning of the primer-terminus between the polymerization and editing active sites (Rodríguez et al., 2009), a result that led us to suggest that in 29 DNAP the primer-terminus switches between both active sites by a passive diffusion mechanism. In this sense, the recent use of single-molecule manipulation method has made possible the study of the dynamics of the partitioning mechanism by applying different tension to a processive single 29 DNAP-DNA complex (Ibarra et al., 2009). Thus, the application of mechanical force to the template causes the gradual intramolecular switch of the primer between the active sites of the protein by decreasing the affinity of the polymerization active site for the template strand with the further disruption of the dsDNA primer-template structure that provokes a fraying of 4-5 bp of dsDNA, allowing primerterminus to reach the exonuclease active site intramolecularly (Ibarra et al., 2009), supporting the passive diffusion mechanism. The energetically unfavorable gradual melting of 4-5 bp of dsDNA should be progressively balanced by the establishment of new and specific interactions with DNA ligands of the thumb subdomain (Pérez-Arnaiz et al., 2006). Such contacts would also guide the primer-terminus to interact with ssDNA ligands of the exonuclease domain that stabilize the primer-terminus at the exonuclease site (de Vega et al., 1996(de Vega et al., , 1998bKamtekar et al., 2004;Pérez-Arnaiz et al., 2006;Rodríguez et al., 2009). Recent development of a single-molecule approach using a nanoscale pore has allowed to conclude that transfer of the primer strand from the polymerase to the exonuclease site takes place before translocation, the pre-translocation state being therefore the branchpoint between the DNA synthesis and editing pathways (Dahl et al., 2014). Once the 3 ′ terminal nucleotide is released, the primer-terminus goes back to the polymerase site and pairs with the template strand in the posttranslocation state being poised to bind the incoming dNTP and resume DNA synthesis (Dahl et al., 2014).

Biotechnological Applications of 29 DNA Polymerase
The two distinctive features of 29 DNAP, high processivity, and strand displacement capacity, together with a remarkably faithful replication, contributed by a high nucleotide insertion fidelity, and an intrinsic proofreading activity, led to the development of isothermal multiple displacement amplification (MDA) currently exploited (Dean et al., 2001(Dean et al., , 2002. These amplification methods based on 29 DNAP show two main advantages respect to classical PCR DNA amplification: first, the use of random hexamer primers eliminates the previous sequence information requirement allowing the amplification of any DNA molecule, and second, the products of the amplification reaction can be much larger than those obtained by classical PCR. In addition, the capacity displayed by 29 DNAP to use circular multiply primed ssDNA templates gave rise to the development of the multiply primed rolling circle amplification of circular DNAs of variable size (Dean et al., 2001). This technology has been successfully exploited to amplify and detect circular viral genomes (Johne et al., 2009), to genotype single nucleotide polymorphisms (Qi et al., 2001), to analyze the genome of non-cultivable viruses (Johne et al., 2009), to detect and identify circular plasmids in zoonotic pathogens (Xu et al., 2008), and to describe new metagenomes (López-Bueno et al., 2009). Recently, we have been able to improve isothermal MDA by making new variants of 29 DNAP . Thus, we have fused DNA binding domains (Helix-hairpin-Helix) to the C-terminus of the polymerase increasing the DNA binding ability of the enzyme without compromising its replication rate. As a result, the new variants display an improved DNA amplification efficiency on both circular plasmids and genomic DNA and are the only 29 DNAP variants with enhanced amplification performance so far.

INITIATION OPPOSITE AN INTERNAL TEMPLATING NUCLEOTIDE: A SMART SOLUTION TO PRESERVE THE FIDELITY DURING INITIATION
The 29 TP/DNAP heterodimer recognizes the replication origins at the genome ends (see Figure 1). Such origins are constituted by specific sequences as well as by the parental TP, the major signal in the template for recognition, a fact that suggests that the heterodimer is recruited to the origin through interactions with the parental TP. The use of heterologous systems in which DNA polymerase, primer TP, and TP-DNA were from the 29 and Nf related phages allowed us to infer specific contacts between the DNA polymerase and the parental TP, as the initiation only occurred when the polymerase and the TP-DNA were from the same phage (González-Huici et al., 2000b;Pérez-Arnáiz et al., 2007). In addition, the presence of mutations in the intermediate domain of both the parental and primer TPs precluded DNA replication, suggesting also a role for the primer TP in the specific recognition of the replication origins (Illana et al., 1998;Serna-Rico et al., 2000;del Prado et al., 2012).
As already indicated, the DNA ends of 29 have a repetition of three nucleotides (3 ′ -TTT. . . . 5 ′ ). Once the replication origins are specifically recognized by the TP/DNA polymerase heterodimer Freire et al., 1996;González-Huici et al., 2000a,b;Pérez-Arnáiz et al., 2007), the DNA polymerase catalyzes the formation of a phosphoester bond between the initial dAMP and the hydroxyl group of Ser232 of the TP. Therefore, during the initiation reaction, the priming Ser232 of the TP is placed at the catalytic site of the DNA polymerase to attack nucleophilically the α-phosphate of the initial dAMP which is inserted opposite the 3 ′ second nucleotide of the template strand (Méndez et al., 1992, see Figure 6A). This reaction is carried out by the catalytic residues responsible for canonical polymerization Salas, 1995, 1996). The initiation reaction implies that the 3 ′ end of the template strand should enter deep into the catalytic site of the DNA polymerase to place the penultimate 3 ′ dTMP of the template strand at the catalytic site (see Figures 6A,B). The interchanging of the priming domains of the related 29 and Nf TPs, allowed us to conclude that this domain is the one responsible for dictating the internal 3 ′ nucleotide used as template during initiation, the 2nd and 3rd in 29 and Nf DNA, respectively (Longás et al., 2008). Recently, we have shown that the aromatic residue Phe230 of the 29 TP priming loop is the one responsible for positioning the penultimate nucleotide at the polymerization site to direct insertion of the initial dAMP during the initiation reaction, most probably by interacting with the 3 ′ terminal base, limiting the internalization of the template strand (see del Prado et al., 2015; Figure 6B). To perform TP-DNA fulllength synthesis, the TP-dAMP initiation product translocates backwards one position to recover the template information corresponding to the first 3 ′ -T, the so-called sliding-back mechanism that requires a terminal repetition of 2 bp. This reiteration permits, prior to DNA elongation, the asymmetric translocation of the initiation product, TP-dAMP, to be paired with the first T residue (Méndez et al., 1992) (see scheme in Figure 7).
We have shown that the sliding-back, or variations of it, is a mechanism shared by the protein-priming systems to restore full-length DNA. In the case of the 29-related phage GA-1, initiation takes place at the 3 ′ second nucleotide of the template (3 ′ -TTT) (Illana et al., 1996). The 29-related phage Nf and the S. pneumoniae phage Cp-1 initiate opposite the 3 ′ third nucleotide of their terminal repetition (3 ′ -TTT) (Martín et al., 1996;Longás et al., 2008), whereas the E. coli phage PRD1 initiates at the fourth nucleotide (3 ′ -CCCC) (Caldentey et al., 1993), being required two and three consecutive sliding-back steps, respectively, to recover the information of the DNA termini (stepwise sliding-back). The case of adenovirus is a little more complex as its genome ends have a duplication of the sequence GTA (3 ′ -GTAGTA). In this virus, the 3 ′ fourth to sixth template positions guides the formation of the TP-CAT initiation product that jumps back to pair with the terminal GTA, a variation of the sliding-back mechanism called jumping-back (King and van der Vliet, 1994) (see scheme in Figure 7).
What is the rationale of the sliding-back mechanism? 29 protein-primed initiation is an unfaithful reaction with a nucleotide insertion discrimination factor about 10 2 . In addition, the 3 ′ -5 ′ exonucleae activity of 29 DNAP cannot release a wrong nucleotide that had been added during the initiation reaction (Esteban et al., 1993). Therefore, the sliding-back mechanism could guarantee the fidelity during the initiation stage through different base pairing checking steps before further elongation of the TP-dNMP complex occurs (Méndez et al., 1992;King and van der Vliet, 1994). Thus, an erroneous TP-dNMP complex will not pair with the terminal 3 ′ -T of the template after the sliding-back, hindering its further elongation. In addition, if an incorrect TP-dNMP product were elongated the resulting TP-DNA molecule could not be used as a template in the next replication round, as the 3 ′ terminus of the template strand would not include the required nucleotide reiteration. The presence of sequence repetitions at the ends of other TP-containing genomes allows to surmise that the sliding-back type of mechanism could be a common feature of protein-primed replication systems (Méndez et al., 1992).

TRANSITION FROM PROTEIN-PRIMED TO DNA-PRIMED REPLICATION
Previous biochemical studies showed that once the initiation reaction has taken place the polymerase incorporates the next 4 nucleotides to the TP-dAMP product while is still complexed with the primer TP (initiation mode), goes through some structural changes during insertion of the sixth to ninth nucleotide (transition mode) and finally dissociates from the primer TP once the tenth nucleotide is added to the growing strand (elongation mode) (Méndez et al., 1997). Resolution of the 29 DNAP/TP complex has given the insights on the transition mechanism, explaining how the polymerase can insert up to nine nucleotides while complexed to the TP (Kamtekar et al., 2006). The transition stage relies on a different strength interaction of the TP priming and intermediate domains with the DNA polymerase (Pérez-Arnáiz et al., 2007). On the one hand, the TP intermediate domain remains in a fixed orientation on the polymerase during insertion of 6-7 nucleotides by means of stable contacts with the TPR1 subdomain. During this stage the weakness of the interaction between the DNA polymerase and the TP priming domain allows the latter to rotate as the DNA is synthesized. The rotation of the TP priming domain with respect to the fixed TP intermediate domain is possible due to the flexibility of the hinge region that connects both domains. Once 6-7 nucleotides have been added, the proximity of the priming Ser to the hinge region would impede a further priming domain FIGURE 7 | Sliding-back (jumping-back) model for the transition from initiation to elongation. TP is represented as a pink oval and DNA polymerase as a gray square. The internal template nucleotide that directs the insertion of the initial nucleotide is shown in bold red letter. Yellow box represents the catalytic active site of the DNA polymerase. Reproduced from de Vega and  rotation, causing heterodimer dissociation (Kamtekar et al., 2006; see Figure 8).

PROTEIN P5, THE VIRAL SINGLE-STRANDED DNA BINDING PROTEIN Structural and Functional Characteristics
Single-stranded DNA-binding proteins (SSBs) are common in all three branches of organisms and in viruses and bind with high affinity to single-stranded (ss) DNA, playing essential roles as accessory proteins in DNA replication, recombination, and repair processes that entail the exposure of ssDNA. SSBs usually bind non-specifically to DNA and can saturate long stretches of ssDNA, thus providing protection against nuclease attack, and preventing the formation of secondary structures (Chase and Williams, 1986;Kur et al., 2005). Furthermore, SSB proteins are involved in specific interactions with several proteins that play important roles in nucleic acids metabolism (Shereda et al., 2008). As a result of these properties, SSBs increase the efficiency and fidelity of a number of DNA amplification methods (Rapley, 1994;Perales et al., 2003;Inoue et al., 2006;Mikawa et al., 2009;Ducani et al., 2014).
From a structural viewpoint, SSBs exist as monomeric or multimeric proteins and, with few exceptions, they share a structural domain named OB-fold (oligonucleotide/ oligosaccharide binding-fold) involved in nucleic acid recognition (Theobald et al., 2003;Savvides et al., 2004). The OB-fold structural domain consists in a close or semi-open beta barrel made out of five-stranded β-strands and a α-helix, commonly between the third and four strands (Murzin, 1993). 29 protein p5 is a single-stranded DNA binding protein (Martin et al., 1989) that protects DNA from nucleases (Martin et al., 1989) and prevents unproductive binding of 29 DNAP to ssDNA generated during replication (Gutiérrez et al., 1991). 29 SSB has high sequence similarity with SSBs from the related podoviruses Nf and GA-1, although 29 and Nf are monomeric in solution, whereas GA-1 SSB is hexameric (Soengas et al., 1995;Gascón et al., 2000a), by means of a N-terminal additional motif (Gascón et al., 2002). Podoviral SSBs share some key hydrophobic residues with unrelated viral SSBs (Gutiérrez et al., 1991) and, indeed, they may also share the SSBs common OBfold, as found by secondary structure prediction and multiple sequence alignment (Figure 9). In agreement with this predicted protein folding, previous circular dichroism spectra indicated that 29 SSB is largely made up of β-strands (Soengas et al., 1997a).
The interaction of 29 SSB with ssDNA is consistent with a moderate cooperative binding to 3-4 nt per molecule, not impaired by ionic conditions . Detailed analysis of intrinsic tyrosine fluorescence quenching upon binding to ssDNA and site-directed mutagenesis indicated that Tyr50, Tyr57, and Tyr76 play essential role in complex formation with DNA (Soengas, 1996;Soengas et al., 1997b).
As other SSBs, 29, Nf, and GA-1 SSBs, are able to unwind duplex DNA (Soengas et al., 1995;Gascón et al., 2000b), suggesting that they can favor DNA replication by unwinding the secondary structures formed in the ssDNA produced during genome replication. However, although all the three SSBs increase DNA replication efficiency (Martin et al., 1989), only 29 SSB enhances the replication rate of the DNA polymerase, especially when strand displacement is impaired, although it does not seem to have specificity for its cognate DNA polymerase (Soengas et al., 1995;Gascón et al., 2000b). Therefore, rather than the interaction of the SSB and its own DNA polymerase, improvement of the replication rate by 29 SSB is mediated by its dynamic dissociation from the nucleoprotein complexes ahead the polymerase, in agreement with its relative low intrinsic binding constant Gascón et al., 2000a).

Biological Role
SSBs are required in stoichiometric quantities with respect to the template rather than in catalytic amounts. Accordingly, 29 SSB is required in high amounts for in vitro 29 genome amplification  and it is an extremely abundant protein in the infected B. subtilis cells (∼700,000 molecules per cell, Martin et al., 1989). Early FIGURE 9 | Multiple sequence alignment of diverse SSBs from prokaryotic origin. Source and GenBank identification number (GI) of each protein is indicated. Alignment was made with Promals3D (Pei et al., 2008), based secondary structure predictions and the crystal structure of E. coli and T7 SSBs (1SRU and 1JE5, respectively, in Protein Data Bank). The protein sequences are colored according to actual or predicted secondary structures (red: alpha-helix, blue: beta-strand). Also, the consensus five beta-strands and the alpha-helix that correspond with a common OB-fold are depicted above the sequences. Note that in the case of T7 SSB the α-helix is between the second and third strands. The last line in each block (Consensus_aa) shows consensus amino acid sequence as follows: genetic characterization of 29 mutants allowed the mapping of temperature-sensitive mutants in gene 5 . Those mutants had a strong impairment in DNA synthesis (Talavera et al., 1972), indicating an essential role in replication of the viral genome that, as mentioned above, was subsequently demonstrated thanks to the in vitro characterization of TP-DNA replication.
Strikingly, recent isolation of a non-sense mutant in gene 5 that only had a 20% reduction in viral yield (Tone et al., 2012), suggested that 29 SSB might be dispensable for viral replication, although it seems to be required in a temperature dependent fashion. These results led the authors to speculate that a host SSB could be able to partially complement the absence of viral SSB at permissive temperatures. However, the molecular mechanism of this possible temperature-dependent role of 29 SB remains unclear.

A HISTONE-LIKE PROTEIN ENCODED BY BACTERIOPHAGE 29
Structural Characteristics and DNA Binding Mechanism The viral protein p6 is a DNA binding protein (DBP) involved both in DNA replication, activating the proteinprimed initiation step, and transcriptional control, modulating the early-late switch (for a detailed review see González-Huici et al., 2004a). This pleiotropic effect is consequence of its role as an architectural protein that organizes and compacts the viral genome, analogously to eukaryotic histones (Serrano et al., 1994).
Structure-function work on p6 indicated that the N-terminal region of the protein plays a role in both DNA binding (Otero et al., 1990) and dimer formation (Abril et al., 2000). By sitedirected mutagenesis, it could be disclosed that residues Ile8 and Val44 are directly involved in protein dimer formation (Abril et al., 2000(Abril et al., , 2002, whereas Lys2, Lys10 and, especially, Arg6, are essential for DNA binding in vitro and viral DNA synthesis in vivo (Bravo et al., 1994b;Freire et al., 1994).
According to footprinting assays (Prieto et al., 1988;Serrano et al., 1990), p6 binding to DNA gives rise to a nucleoprotein complex formed by a repeated motif of p6 dimers bound to a 24 bp DNA segment. Thus, a protein monomer would contact and bend the DNA every 12 bp, suggesting a model in which the DNA would wrap around a multimeric core of protein p6, forming a right-handed superhelix that comprises around 63 bp per turn (Serrano et al., 1990(Serrano et al., , 1993a. As a consequence of DNA wrapping, this nucleoprotein complexes show a remarkable reduction in length with respect to naked DNA, between 4.2-to 6.5-fold (Serrano et al., 1993a;Gutiérrez et al., 1994).
In vivo, p6 is able to discriminate between bacterial and viral DNA by their different superhelicity (González-Huici et al., 2004b). Thus, p6 is able to restrain positive supercoiling of the DNA in vitro (Prieto et al., 1988;Serrano et al., 1993b) and binds all along 29 DNA in vivo with a much higher affinity than for plasmid DNA, although binding to plasmid DNA is enhanced by decreasing the negative supercoiling (González-Huici et al., 2004c). Thus, the presumably lower negative superhelicity of 29 DNA respect to host chromosome likely makes the viral genome an appropriate target for the binding of p6 (Serrano et al., 1994;González-Huici et al., 2004b). Interestingly, the preferential binding of 29 p6 to the lower negatively supercoiled viral genome seems to be quite specific, since GA-1 p6, which has a highly conserved sequence (58% similarity, 39% identity), does not show this binding pattern (Freire et al., 1996) and accordingly, GA-1 p6 complex with 29 DNA is not functional (Alcorlo et al., 2007).
Moreover, p6 has a binding specificity to the ends of the 29 linear genome, which has a key role in the initiation step of replication (see below). This binding occurs at recognition regions that were mapped between positions 62-125 from the right end, and between positions 46-68 from the left one (Serrano et al., 1989). However, p6 does not recognize a sequence signal, but rather a sequence-dependent bendability pattern present in the recognition sites that act as a nucleation site for protein p6/DNA complex formation (Serrano et al., 1993a;González-Huici et al., 2004b).

Functional Implications of p6 Nucleoprotein Complex
Protein p6 is essential for 29 DNA replication in vivo (Carrascosa et al., 1976;Bravo et al., 1994b). In vitro, p6 stimulates initiation as well as the transition to elongation (Pastrana et al., 1985;Blanco et al., , 1988. Initiation activation requires the formation of the protein p6 complex with 29 DNA terminal fragments (Serrano et al., 1989) and it was suggested to undergo through transient unwinding of DNA at the p6 specific binding sites that would favor interaction of the TP/DNAP complex with the template strand (Serrano et al., 1993b). In line with this hypothesis, 29 origins with partially unpaired ends showed increased utilization (up to 30-fold respect to wild type origins) (Gella et al., 2014). Initiation of these modified origins was still stimulated by p6, although to a lesser extent (around 1.5-fold) than the wild type origin (2.8-fold).
As mentioned above, p6 is also important for the control of transcription, either by itself or together with the viral transcriptional regulator p4 Salas, 2000, 2001). Thus, protein p6 switches off very early transcription from promoter C2, as shown by in vivo and in vitro studies, impairing the RNA polymerase complex access to the nucleoprotein complex at the promoter region (Serrano et al., 1989;Camacho and Salas, 2001). Moreover, formation of p6 nucleoprotein complex promotes p4-mediated repression of promoters A2b and A2c and activation of the A3 promoter (Calles et al., 2002).
In the context of the infected cell, p6 is highly abundant, which would favor oligomerization and formation of p6 nucleocomplex (Abril et al., 1997), which might be even more favored under the crowded intracellular environment (Alcorlo et al., 2009). This high density is in agreement with a histone-like function that would complex with the entire genome (Serrano et al., 1994;Holguera et al., 2012), analogously to cellular histones. At early infection stages, p6 localizes mainly in a peripheral helix-like configuration , whereas the viral genome and the replication machinery associates with the host nucleoid (Muñoz-Espín et al., 2010). Since protein p6 is essential to initiate 29 DNA replication, it was suggested that a small amount of protein p6 (undetectable by immunofluorescence) would be recruited early at the bacterial nucleoid, establishing the appropriate conditions at the phage DNA ends to achieve the first rounds of replication. Then, and as viral DNA replication progresses, p6 is recruited to the bacterial nucleoid and, by topological recognition of the 29 DNA, avoids its sequestration by the higher volume of the bacterial DNA. Under this scenario, 29 p6 constitutes a histone-like protein specific for the viral genome, whose temporal and spatial subcellular localization is determined by its essential roles in genome replication and transcription .

ROLE OF HOST DNA-BINDING PROTEINS IN 29 DNA REPLICATION
Bacteriophages have developed different strategies to inactivate or take advantage of cellular enzymes in their own benefit (Roucourt and Lavigne, 2009). During 29 infection several B. subtilis DNA-binding proteins have been shown to play a role in the development of the infective cycle.

DNA Gyrase
Chromosomal DNA topology is controlled by various hostencoded topoisomerases, such as DNA gyrase (topoisomerase II) (Drlica, 1992). Despite containing a TP covalently linked to the 5 ′ ends and therefore not being covalently closed, 29 DNA is topologically constrained in vivo (González-Huici et al., 2004c). In this sense, it has been shown that the gyrase inhibitor novobiocin but not nalidixic acid, which also inhibits DNA gyrase but does not have topological effects on DNA, increases the binding of protein p6 to the viral genome in vivo. In addition, both novobiocin and nalidixic acid impair viral DNA replication in vivo, suggesting that B. subtilis gyrase is involved in viral DNA replication (González-Huici et al., 2004c). A topologically constrained DNA should be allowed to rotate freely during the replication process, explaining the necessity of the bacterial DNA gyrase . Moreover, 29 genome possesses two convergently oriented transcription units encompassing genes 7-16 and 17-16.5. Hence, without the action of DNA gyrase, a highly positive supercoiled region would be generated between the two convergently oriented transcription units, preventing the advance of the RNA polymerase, and/or inducing DNA polymerase template switching when encountering this blockage. In fact, during 29 infection subgenomic viral DNA molecules ranging from 1 to 8 Kb are accumulated, originated mainly from the right end of the genome and that these kind of molecules do not accumulate when B. subtilis cells are infected with the transcription deficient mutant 29 sus4(56), which does not express protein p4 (Murthy et al., 1998).
The topological constraint of bacteriophage 29 genome could be achieved by binding of the parental TPs either directly to the nucleoid or to the bacterial membrane through the interaction with other viral proteins such as p1 or p16.7 (see above) (Bravo et al., 2000;Serna-Rico et al., 2003;Muñoz-Espín et al., 2010).

Uracil-DNA Glycosylase
A potential threat to genome integrity is the presence of uracil residues in DNA. Uracil is eliminated from DNA genomes by the base excision repair pathway (BER), which is initiated with the enzymatic activity of a uracil-DNA glycosylase (UDG). These enzymes (Family 1) selectively remove uracil bases from both single-and double-stranded DNA, cleaving the N-glycosidic bond between the base and the deoxyribose, leaving therefore an abasic site . This abasic site is then repaired through the sequential action of an apurinic/apyrimidinic endonuclease, DNA polymerase and DNA ligase. Most eukaryotic and prokaryotic cells encode a UDG to maintain the integrity of DNA genomes. However, there are some cases in which the presence of uracil in DNA could be desirable. For instance, B. subtilis phage PBS1 (and its clear-plaque isotype PBS2) genome contains uracil instead of thymine and, consequently, encodes a UDG inhibitor (called Ugi) to assure an efficient viral genome replication (Katz et al., 1976;Cone et al., 1980;. Additionally, phage T5 infection induces an inhibitor of E. coli UDG that has not yet been identified (Warner et al., 1980). Interestingly, despite having a non-uracil containing genome, phage 29 encodes a UDG inhibitor, a small acidic protein of 56 amino acids called p56 (Serrano-Heras et al., 2006). Protein p56 is expressed early after infection and interacts with B. subtilis UDG, inhibiting its activity (Serrano-Heras et al., 2006). In vitro experiments showed that protein p56 blocks the DNAbinding ability of UDG, and structural data suggest that it does it by mimicking the structure of DNA (Serrano-Heras et al., 2007;Asensio et al., 2011;Baños-Sanz et al., 2013;Cole et al., 2013). As mentioned above, the mechanism of 29 DNA replication involves the generation of replicative intermediates that contain large stretches of ssDNA (Harding and Ito, 1980;Inciarte et al., 1980; see Figure 1). If uracil residues were present in these stretches of ssDNA by either the misincorporation of deoxyuridine monophosphate (dUMP) during the replication process or by the spontaneous deamination of cytosine in DNA, the elimination of these lesions by the BER pathway would give rise to the loss of terminal viral DNA regions. In fact, it has been shown that 29 DNA polymerase can incorporate dUMP during DNA synthesis with a catalytic efficiency of only 2-fold lower than dTMP, and perform the extension of base-paired uracil residues to give full-length DNA in vitro (Serrano-Heras et al., 2008). Hence, by encoding an UDG inhibitor, 29 prevents the elimination of uracil residues that could be present in the ssDNA portions of the genome replicative intermediates and that would compromise viral genome integrity (Serrano-Heras et al., 2006;Muñoz-Espín et al., 2012).
It is worth mentioning that 29-related phages PZA, B103, Nf, and GA-1 encode homologs of p56. The product of GA-1 gene 56 was purified and shown to inhibit UDG activity in extracts of both B. subtilis and B. pumilus, which is the natural host of GA-1 .
The elucidation of the function of several 29 proteins yet to be characterized and the improvement of in vivo techniques for both protein-protein and protein-DNA interactions detection will lead to a better understanding of the virus-host interactome in the future.

AUTHOR CONTRIBUTIONS
All authors listed have made substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
This work has been supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2014-52656-P to MS) and (BFU2014-53791-P to MV), ComFuturo Grant from Fundación General CSIC (to MR) and by an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular "Severo Ochoa."