DNA: Natural Single‐stranded

Abstract

Single‐stranded circular or linear viral genomes have been used as model systems to investigate the mechanisms of DNA replication and gene expression and for the development of highly versatile cloning vectors. Flexible single‐stranded DNA can form hairpin/secondary structures by base pairing between regions of intramolecular sequence complementarity. Such structures are readily detected and appear to play important roles in virus‐associated functions in vivo.

Keywords: single‐stranded DNA; phages; replicative form; secondary structure; overlapping genes

Figure 1.

(a) Isometric bacteriophage ØX174 and a schematic representation of the phage icosahedron. A spike made of gene H and gene G proteins is located in the diagram of the virion at the apex (lightly shaded) and is surrounded by gene F coat proteins (dark shaded area). (b) Filamentous bacteriophage M13. (c) Circular single‐stranded DNA of phage MAC‐1. Adapted from Kornberg and Baker and Roberts et al.

Figure 2.

(a) Secondary structure of single‐stranded DNA from MAC‐1 phage. Phage DNA was subjected to electrophoresis in agarose gel (1%) and visualized after staining with ethidium bromide. Lane 1 shows standard pBR322 plasmid DNA containing supercoiled (I), circular (II), and linear (III) DNA; lane 2, unincubated MAC‐1 DNA; lane 3, MAC‐1 DNA incubated at room temperature for 1 h before electrophoresis; lane 4, DNA in lane 3 was heated to 95°C and then quickly cooled on ice before being subjected to electrophoresis. Arrow indicates the position of base‐paired MAC‐1 DNA dimers. (b) Digestion of MAC‐1 single‐stranded DNA with restriction enzyme HaeIII. MAC‐1 phage DNA was incubated at 37°C without or with restriction enzyme HaeIII for 15, 30 or 60 min. An aliquot was removed and subjected to electrophoresis in agarose gel (2.3%). Lane 1, MAC‐1 DNA (resolved into monomer and dimer) at 0 time; lane 2, MAC‐1 DNA incubated without enzyme for 30 min; lanes, 3, 4 and 5 MAC‐1 DNA incubated with HaeIII for 15, 30 and 60 min, respectively.

Figure 3.

Secondary structure of MAC‐1 single‐stranded DNA . DNA sequence ladder from Bdellovibrio bacteriovorus single‐stranded phage MAC‐1 was generated by using either Sequenase Ver. 2 protocol at 37°C (b) or ΔTaq DNA polymerase protocol at 60°C (c). Gel loading order in each case from left to right was GATC (Ranu, ). Arrows on right of (b) indicate full‐stops, with the initial full‐stop indicated by a bold arrow. Schematic representation shows secondary structures of MAC‐1 ssDNA based on data from the sequence ladder generated using Sequenase Ver. 2 or ΔTaq DNA polymerase. Potential secondary structures that lead to formation of DNA polymerase full‐stops are indicated by arrows (a).

Figure 4.

Hairpin/secondary structure of single‐stranded DNA sequence in G4 and ØX174. Possible hairpin/secondary structures (I and II) of DNA sequences at the complementary‐strand origin of replication (ori) of phage G4 and the hairpin structure of the sequence recognition site of protein n′ possibly involved in DNA replication of ØX174 are shown. Numbers indicate nucleotide positions on ØX174 map. Adapted from Baas and Jansz .

Figure 5.

Restriction endonuclease map of MAC‐1 phage DNA (a) and genetic map of ØX174 with function of gene products (b). Arrows indicate the positions of the three promoters and the direction of transcription. Adapted from Kornberg and Baker . (c) A segment of DNA (and amino acid) sequence of ØX174 showing overlap of genes A, B and K.

Figure 6.

Schematic diagram showing three stages of ØX174 DNA replication. SSB, single‐stranded DNA ‐binding protein. Adapted from Baas and Jansz , and Kornberg and Baker .

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References

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Further Reading

Berns KI (1996) Parvoviridae: the viruses and their replication. In: Fields BN, Knipe DM and Howley PM (eds) Fields’ Virology, pp. 2173–2198. Philadelphia: Lippincott‐Raven.

Hayashi MN, Aoyama A, Richardson DL and Hayashi M (1988) Biology of the bacteriophage ØX174. In: Calendar R (ed.) The Bacteriophages, pp. 1–71. New York: Plenum Press.

Kodaira K‐I, Godson NG and Taketo A (1995) Comparative studies on the minus origin mutant of Escherichia coli spherical single‐stranded DNA phages. Biochimica et Biophysica Acta 1260: 191–199.

Messing J (1996) Cloning single‐stranded DNA. Molecular Biotechnology 5: 39–47.

Shaw JG (1996) Plant viruses. In: Fields BN, Knipe DM and Howley PM (eds) Fields’ Virology, pp. 499–532. Philadelphia: Lippincott‐Raven.

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Ranu, Rajinder S(Apr 2001) DNA: Natural Single‐stranded. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0001338]