Replication and Transcription of Bacteriophage ϕ29 DNA

Margarita Salas; Fernando Rojo

doi:10.1128/9781555818388.ch58

Chapter 58 : Replication and Transcription of Bacteriophage ϕ29 DNA

Authors: Margarita Salas¹, Fernando Rojo¹

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Affiliations: 1: Centro de Biologia Molecular (CSIC-UAM), Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain

Content Type: Monograph

Publication Year: 1993

Category: Bacterial Pathogenesis; Microbial Genetics and Molecular Biology

Book DOI: 10.1128/9781555818388

Chapter DOI: 10.1128/9781555818388.ch58

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Abstract:

The genome of Bacillus subtilis phage ø29 consists of a linear, double-stranded DNA 19,285 bp long with a 6-bp-long inverted terminal repeat (AAAGTA; 32, 114) and a terminal protein (TP) covalently linked at the 5' ends. Viral genes 1, 2, 3, 5, 6, and 17 are required for ø29 DNA replication. ø29 DNA polymerase is inhibited by drugs that are known inhibitors of eukaryotic DNA polymerase α such as aphidicolin, phosphonoacetic acid, and the nucleotide analogs butyl-anilino dATP (BuAdATP) and butyl-phenyl dGTP (BuPdGTP). The amino acid sequence RGD, which is found in cell adhesion proteins, is present at positions 256 to 258 in the ø29 and M2 TPs. Activation of the initiation of ø29 DNA replication by p6 requires not only formation of the complex but also its correct positioning relative to the ø29 DNA ends, suggesting that other proteins involved in the initiation of ø29 DNA replication (TP and/or DNA polymerase) recognize p6 at a precise position. Transcription of the ø29 genome takes place in two stages. At the beginning of the infection only the genes involved in DNA replication and transcription regulation are expressed. Genes coding for structural components of the phage particle and for proteins involved in morphogenesis and cell lysis are expressed later on in infection.

Citation: Salas M, Rojo F. 1993. Replication and Transcription of Bacteriophage ϕ29 DNA, p 843-857. In Sonenshein A, Hoch J, Losick R (ed), Bacillus subtilis and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch58

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Replication and Transcription of Bacteriophage ϕ29 DNA

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Figure 1

Genetic and transcriptional maps of phage ϕ29 genome. (A) Genetic map (adapted from reference 69 ). Arrows indicate directions of transcription. The TP is shown attached to the 5' ends of the genome. (B) Transcription map. Arrowheads indicate directions of transcription from the different promoters (see text). The region containing the main early and late promoters is magnified to show the relative position of each promoter. Transcription terminators (TA1, TB1, TB2, and TD1) are represented by thick, filled arrowheads. The position and approximate length of each gene is indicated. SSB, single-stranded-DNA-binding protein.

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Figure 1

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Figure 2

Different stages and viral gene products involved in ϕ29 DNA replication. Based on structural and functional studies (see text for details), a two-domain structure analogous to that of the Klenow fragment of polymerase I is extrapolated for ϕ29 DNA polymerase. ϕ29 primer TP is indicated by black, and parental TP is shaded. DBP, DNA-binding protein; SSB, single-stranded-DNA-binding protein.

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Figure 2

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Figure 3

Sliding-back model for transition from protein-primed initiation to DNA elongation. Darkly shaded areas correspond to polymerization domains of ϕ29 DNA polymerase that define a cleft proposed to be used both as DNA- and TP-binding sites. ϕ29 TP, serving as primer for the nascent strand, is indicated by black, whereas parental ϕ29 TP, which is covalently bound to the displaced strand, is indicated by light shading. The sequence indicated corresponds to the 6-bp inverted terminal repeat present at each ϕ29 DNA end. See text for details.

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Figure 3

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Figure 4

p4-binding site. The DNA region represented spans the area from the early A2b promoter (bottom left) to the divergent late A3 promoter (right), with a p4-binding site between them. RNA polymerase (RNP) is bound to the late A3 promoter. Positions protected from the attack of hydroxyl radicals by p4 or by the RNA polymerase bound at the late A3 promoter are shown by open and filled dots, respectively. p4-binding sequences are represented by thickened lines in the DNA sequence. Guanine residues whose methylation interferes with p4 binding to DNA are also shown (©)· Arrows indicate positions that become hypersensitive to DNase I cleavage upon p4 binding, most likely as a consequence of the protein-induced DNA curvature. Positions -56 and -102 do not appear when both p4 and RNA polymerase are bound to the late A3 promoter and are shown in parentheses. Note that the p4-binding site partially overlaps the early A2b promoter, thus displacing the RNA polymerase from it and directing the polymerase to the late A3 promoter.

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Figure 4

References

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