Lagging strand replication of rolling-circle plasmids in Streptomyces lividans: an RNA polymerase-independent primer synthesis
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The rolling circle (RC) mechanism of DNA replication generating single-stranded DNA (ssDNA) intermediates is common in various high-copy circular plasmids in Streptomyces, and the ssDNA released after leading strand synthesis is converted to its double-stranded form (dsDNA) by the host proteins. The in vivo and in vitro lagging strand syntheses from ssDNA replicative intermediates of RC plasmid pSN22 in Streptomyces lividans was characterized. The presence or absence of the single-strand origin (sso), the replication initiation site of lagging strand synthesis, did not significantly affect the copy numbers of pSN22 derivatives. In vivo lagging strand synthesis was not affected by the rifampicin inhibition of S. lividans RNA polymerase. Likewise, in vitro lagging strand synthesis using cell-free extracts revealedsso-independent, rifampicin-resistant lagging strand synthesis in S. lividans. Although all four dNTPs are usually required for the initiation of such synthesis, the presence of only one NTP was sufficient to carry outlagging strand synthesis in vitro. Interestingly, the cell-free extract of exponential-phase cells required less ATP than that of stationary-phase cells. These results reveal a predominant RNA polymerase-independent priming system in S. lividans that may be a result of the stabilization of RC plasmids lacking sso in S. lividans.
KeywordsReplication Streptomyces Priming Lagging strand
We greatly thank to Drs. T. Kieser, H. Araki and T. Ito for valuable discussions; Dr E. Ko-Mitamura for editorial suggestions and manuscript corrections; and Dr H. Masai for valuable comments on the general priming of E. coli.
- Bruand C, Ehrlich SD, Jannière L (1995) Primosome assembly site in Bacillus subtilis. EMBO J 11:2642–2650Google Scholar
- Conrad SE, Campbell JL (1979) Characterization of an improved in vitro DNA replication system for Escherichia coli plasmids. Nucl Acids Res 6:3289–3303Google Scholar
- Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schrempf H (1985) Genetic manipulation of Streptomyces: a laboratory manual. The John Innes Foundation, Norwich, UKGoogle Scholar
- Jannière L, Gruss A, Ehrlich SD (1993) Plasmids. In: Sonenshein AL, Losick R, Hoch JA (eds) Bacillus subtilis and other gram positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington DC, pp 625–644Google Scholar
- Kornberg A, Baker TA (1992) DNA replication, 2nd edn. WH Freeman & Co, New YorkGoogle Scholar
- Kramer MG, Khan SA, Espinosa M (1997) Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis. EMBO J. 16:5784–5795Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
- Thompson J, Cundliffe E (1981) Purification and properties of an RNA methylase produced by Streptomyces azureus and involved in resistance to thiostrepton. J Gen Microbiol 124:291–297Google Scholar