Role of coliphage M13 gene 5 in single-stranded DNA production

Salstrom, John S.; Pratt, David

doi:10.1016/0022-2836(71)90061-1

Cited by 145 publications

(63 citation statements)

References 19 publications

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“…Only one phage-specified protein, the gene-5 protein, is required for single-strand synthesis (4). This protein plays a negative role, preventing the conversion of newly synthesized viral single strands into double-stranded RF molecules (12 2 Ag/ml of thymidine at 34°, to 1 X 108 cells/ml. 30 ml Of the culture was infected with M 13 at a multiplicity of 100, another 30 ml was used as an uninfected control.…”

Section: Discussionmentioning

confidence: 99%

Replication of Bacteriophage M 13: Inhibition of Single-Strand DNA Synthesis by Rifampicin

Staudenbauer¹,

Hofschneider²

1972

Proc. Natl. Acad. Sci. U.S.A.

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Synthesis of single-stranded DNA of bacteriophage M 13 is blocked by rifampicin. This inhibition is not observed in an Escherichia coli mutant possessing an RNA polymerase resistant to rifampicin. Since rifampicin stops single-strand synthesis faster than chloramphenicol, inhibition by rifampicin does not seem to result from a transcriptional block of protein synthesis, particularly in view of the long half-life of the messenger RNA for M 13. It is, therefore, concluded that the interaction of rifampicin with RNA polymerase directly affects single-strand DNA synthesis. Possible mechanisms are discussed.The DNA of the filamentous phage M 13 replicates in three stages (1, 2). First, the infecting single-stranded DNA is converted by host enzymes into a double-stranded form (SS --RF). Early in the infection the replicative forms multiply to establish a pool of RF molecules (RF --RF). Later, double-strand synthesis ceases almost completely, and the RF molecules serve as precursors for single-strand synthesis (RF SS). Single-stranded viral DNA is formed by an asymmetric replication process in which the viral strand of the RF is displaced as a new one is synthesized (3).These three stages of phage DNA repliction show differences in their sensitivity towards antibiotics. Inhibition of protein synthesis by chloramphenicol has no effect on the SS --RF conversion (4). Ongoing RF replication is also rather insensitive to chloramphenicol, and continues at a slowly decreasing rate after all protein synthesis has stopped (5). If chloramphenicol is added late in the infection, an immediate switch back from single-strand synthesis to RF replication is observed (6). However, the conversion of single-stranded parental DNA into the replicative form, as well as the multiplication of the double-stranded forms, are rapidly inhibited by rifampicin (5), a specific inhibitor of RNA polymerase.Since it has been shown in this lab that the specific messenger RNA for phage M 13 has an exceptionally long halflife (ref. 7 and unpublished data), we expected that blockage of transcription with rifampicin should have a considerably delayed effect on single-strand synthesis and phage maturation, as compared to chloramphenicol treatment. However, after we added rifampicin to M 13-infected cells late in infection, a rapid halt of phage release was observed (Fig. 1). Following up this observation, we found, as shown in this paper, that addition of rifampicin results in an immediate stop of single-strand synthesis. Since no inhibition was observed in an Escherichia coli mutant with an RNA polymerase resistant to rifampicin, we conclude that rifampicin stops M 13 single-strand synthesis by interaction with RNA polymerase. METHODS AND RESULTSThe effect of rifampicin and chloramphenicol on DNA synthesis in uninfected cells and in M 13-infected cells was measured 2 hr after infection, a time when single-strand synthesis is predominant, by exposure of aliquots of the treated and untreated cultures to [3H thymidine for 1 min. As shown in Fig. 2A, ch...

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Section: Discussionmentioning

confidence: 99%

Replication of Bacteriophage M 13: Inhibition of Single-Strand DNA Synthesis by Rifampicin

Staudenbauer¹,

Hofschneider²

1972

Proc. Natl. Acad. Sci. U.S.A.

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“…Late in infection a low but significant amount of radioactive pulse-labeled complementary strands in RF DNA can be detected [133]. The transition from stage II to stage III replication takes place very smoothly and is mainly dependent on the amount of gene V protein present [134][135][136]. A further difference from the isometric phages is the complete separation of the replication process from morphogenesis.…”

Section: Vb Filamentous Phagesmentioning

confidence: 99%

DNA replication of single-stranded Escherichia coli DNA phages

Baas

1985

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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“…The gene V protein from the Ff filamentous bacteriophages (M 13, f l , and fd) plays critical roles in the life cycle of those phages (Salstrom & Pratt, 1971). GVP is a dimeric protein of 87 amino acids ( Fig.…”

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confidence: 99%

Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X‐ray crystallography

Gao

Zhang

et al. 1997

Protein Science

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The high-resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved recently for the wild-type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP-ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4187-197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer-dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X-ray crystallographic analysis at 2.0 8, resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long-range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.

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Role of coliphage M13 gene 5 in single-stranded DNA production

Cited by 145 publications

References 19 publications

Replication of Bacteriophage M 13: Inhibition of Single-Strand DNA Synthesis by Rifampicin

Replication of Bacteriophage M 13: Inhibition of Single-Strand DNA Synthesis by Rifampicin

DNA replication of single-stranded Escherichia coli DNA phages

Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X‐ray crystallography

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