Phage P4 DNA replication<i>in vitro</i>

Orejas, Ramn Díaz; Ziegelin, Günter; Lanka, Erich

doi:10.1093/nar/22.11.2065

Cited by 15 publications

(21 citation statements)

References 32 publications

(21 reference statements)

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“…In vivo it has been shown that the P4 ␣ protein functions in trans at oriII (35). In an in vitro P4 DNA replication system ␣ acts also in trans at oriI (11).…”

Section: Discussionmentioning

confidence: 99%

The repA Gene of the Linear Yersinia enterocolitica Prophage PY54 Functions as a Circular Minimal Replicon in Escherichia coli

et al. 2005

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The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3 portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3 end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3 end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements.Three temperate bacterial viruses of different organisms generating linear prophages are known, PY54, N15, and PKO2. The DNA carries covalently closed hairpins at their ends. Hence we propose the name "hairpin prophage." Two of the phages, PY54 of Yersinia enterocolitica and PKO2 of Klebsiella oxytoca, have been discovered very recently (4, 27). However, the paradigm of the linear prophages, N15 of Escherichia coli, was described already in 1967 by Victor Ravin (13). The linear DNA form is also known from brown algae viruses (7,8) and in the pathogens Borrelia burgdorferi and Agrobacterium tumefaciens (2,14). Although a straightforward mechanistic scheme has been suggested by Rybchin and Svarchevsky (31), the replication of the linear DNA is still poorly understood. The replication model is still valid today. The crucial components are (i) an origin of replication of the theta type proposing bidirectional DNA synthesis, (ii) a multifunctional phage-encoded replication protein guiding the initiation of replication, and (iii) a protelomerase essential for the conversion of circular precursor DNA into the linear form at a sequence called the telomere resolution site. The initial work on the phage systems apparently stimulated studies on the Borrelia system (5).The protelomerase is a tyrosine integrase-like enzyme that catalyzes the intermolecular strand transfer in the double-stranded DNA molecule by cleavage and rejoining of phosphodiester bonds to yield the linear prophage with closed hairpin ends (9, 10). The same principle of action of protelomerases has been confirmed on several enzymes of different origins (4, 16, 21). It was rather simple to predict and locate the telomere resolution sites because they map at the ends of the linear DNA. In the c...

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“…In vivo it has been shown that the P4 ␣ protein functions in trans at oriII (35). In an in vitro P4 DNA replication system ␣ acts also in trans at oriI (11).…”

Section: Discussionmentioning

confidence: 99%

The repA Gene of the Linear Yersinia enterocolitica Prophage PY54 Functions as a Circular Minimal Replicon in Escherichia coli

et al. 2005

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“…To adapt the production of TrxA-Cnr to our convenient p tac /lacI-regulated system in a recA background (30), the trxA-cnr fusion including the cII ribosome binding site (NsiI-BamHI fragment) was inserted into pMS119HE prepared with PstI and BamHI to result in pTZ1106. To facilitate the purification of TrxA-Cnr, we inserted a (His) 6 tag into the active-site loop of the thioredoxin portion of trxA-cnr by using the unique RsrII site of pTZ1106 to give pTZ1106His.…”

Section: Methodsmentioning

confidence: 99%

“…Satellite phage P4 can replicate bidirectionally via the -mode under the control of its own initiator protein (6,15). This protein, called gp␣ (777 amino acids), has DNA binding, helicase, and primase activity (31).…”

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confidence: 99%

Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha

et al. 1997

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Bacteriophage P4 DNA replication depends upon the phage-encoded ␣ protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr). The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr. We searched for P4 mutants that suppressed this phenotype (Cnr resistant [␣cr]). Eight independent mutants that grew in the presence of high levels of Cnr were obtained. None of these can establish the plasmid state. Each of these mutations lies in the DNA binding domain of gp␣ that occupies the C terminus of the protein. Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V. A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of ␣ protein in vitro. The ␣cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein. We present evidence that Cnr protein interacts with the gp␣ domain that binds specifically to DNA and that gp␣cr mutations impair this interaction. We hypothesize that gp␣-Cnr interaction is essential for the control of P4 DNA replication.

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“…The three activities of gp␣ provide the basis of independence of P4 replication from the chromosomally encoded initiation factors and helicase(s) essential in initiation and elongation. From in vitro replication data, it was concluded that the host-specified elongation proteins required for P4 replication include at least DNA polymerase III holoenzyme, SSB, and DNA topoisomerase(s) (13). P4 DNA replication starts bidirectionally at ori and proceeds via -type double-stranded intermediates (13,27).…”

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confidence: 99%

“…From in vitro replication data, it was concluded that the host-specified elongation proteins required for P4 replication include at least DNA polymerase III holoenzyme, SSB, and DNA topoisomerase(s) (13). P4 DNA replication starts bidirectionally at ori and proceeds via -type double-stranded intermediates (13,27). Most likely, both daughter strands, once initiated at ori, are synthesized continuously until the site of replication termination is reached.…”

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confidence: 99%

Domain structure of phage P4 alpha protein deduced by mutational analysis

et al. 1995

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Bacteriophage P4 DNA replication depends on the product of the ␣ gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the ␣ gene. Sequence analysis of the entire gene predicted a domain structure for the ␣ polypeptide chain (777 amino acid residues, M r 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the ␣ polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for ␣ mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.Bacteriophage P4 is a temperate satellite phage of Escherichia coli that relies on morphogenetic and lysis functions of the helper phage P2 for its propagation (30). The P4 prophage state is maintained either by integration into the host chromosome or by autonomous replication as a multicopy plasmid. Lysogenization of the host, DNA replication during the lytic cycle, and establishment of the plasmid state all occur independently of helper phage (30). Replication of P4 DNA requires only the P4 ␣ gene product (gp␣) and two cis-acting elements, namely, the origin of replication ori and the sequence-related region crr (cis replication region) (7,26,30,63). Another P4 gene, cnr (copy number regulation), maps directly upstream of ␣; its product has been suggested to control P4 DNA replication, in particular at the plasmid level, maybe by direct interaction of Cnr with gp␣ (59). Thus, protein-protein interaction might modulate at least one specific function of the ␣ protein.The ␣ protein is a multifunctional phage replication protein that recognizes ori and crr by binding to octamer sequences (type I repeats, TGTTCACC [16]) (64). In addition, gp␣ catalyzes the unwinding of DNA via its helicase activity. DNA is unwound with a 3Ј to 5Ј polarity with respect to the strand that gp␣ has bound. The helicase action is fueled by nucleoside triphosphate (NTP) hydrolysis of an interrelated singlestranded DNA-dependent NTPase. gp␣ also functions as a primase, with a template-dependent oligonucleotide-synthesizing activity (56, 64). The primase activity of gp␣ can be replaced by the primase of the host, the DnaG protein (56). However, the P4 burst sizes in the presence of a primase-null ␣ protein and an active DnaG protein were always small, indicating that DnaG can only partially substitute for the primase fu...

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Phage P4 DNA replicationin vitro

Cited by 15 publications

References 32 publications

The repA Gene of the Linear Yersinia enterocolitica Prophage PY54 Functions as a Circular Minimal Replicon in Escherichia coli

The repA Gene of the Linear Yersinia enterocolitica Prophage PY54 Functions as a Circular Minimal Replicon in Escherichia coli

Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha

Domain structure of phage P4 alpha protein deduced by mutational analysis

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