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. 1991 Apr;127(4):649–655. doi: 10.1093/genetics/127.4.649

Deletions in Plasmid Pbr322: Replication Slippage Involving Leading and Lagging Strands

K Weston-Hafer 1, D E Berg 1
PMCID: PMC1204393  PMID: 2029966

Abstract

We test here whether a class of deletions likely to result from errors during DNA replication arise preferentially during synthesis of either the leading or the lagging DNA strand. Deletions were obtained by reversion of particular insertion mutant alleles of the pBR322 amp gene. The alleles contain insertions of palindromic DNAs bracketed by 9-bp direct repeats of amp sequence; in addition, bp 2 to 5 in one arm of the palindrome form a direct repeat with 4 bp of adjoining amp sequence. Prior work had shown that reversion to Amp(r) results from deletions with endpoints in the 9- or 4-bp repeats, and that the 4-bp repeats are used preferentially because one of them is in the palindrome. To test the role of leading and lagging strand synthesis in deletion formation, we reversed the direction of replication of the amp gene by inverting the pBR322 replication origin, and also constructed new mutant alleles with a 4-bp repeat starting counterclockwise rather than clockwise of the insertion. In both cases the 4-bp repeats were used preferentially as deletion endpoints. A model is presented in which deletions arise during elongation of the strand that copies the palindrome before the adjoining 4-bp repeat, and in which preferential use of the 4-bp repeats independent of the overall direction of replication implies that deletions arise during syntheses of both leading and lagging strands.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albertini A. M., Hofer M., Calos M. P., Miller J. H. On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions. Cell. 1982 Jun;29(2):319–328. doi: 10.1016/0092-8674(82)90148-9. [DOI] [PubMed] [Google Scholar]
  2. Bedinger P., Munn M., Alberts B. M. Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates. J Biol Chem. 1989 Oct 5;264(28):16880–16886. [PubMed] [Google Scholar]
  3. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  4. Chow L. T., Davidson N. Electron microscope study of the structures of lambdadv DNAs. J Mol Biol. 1974 Jun 15;86(1):69–89. doi: 10.1016/s0022-2836(74)80008-2. [DOI] [PubMed] [Google Scholar]
  5. Collins J., Volckaert G., Nevers P. Precise and nearly-precise excision of the symmetrical inverted repeats of Tn5; common features of recA-independent deletion events in Escherichia coli. Gene. 1982 Jul-Aug;19(1):139–146. doi: 10.1016/0378-1119(82)90198-6. [DOI] [PubMed] [Google Scholar]
  6. DasGupta U., Weston-Hafer K., Berg D. E. Local DNA sequence control of deletion formation in Escherichia coli plasmid pBR322. Genetics. 1987 Jan;115(1):41–49. doi: 10.1093/genetics/115.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Farabaugh P. J., Schmeissner U., Hofer M., Miller J. H. Genetic studies of the lac repressor. VII. On the molecular nature of spontaneous hotspots in the lacI gene of Escherichia coli. J Mol Biol. 1978 Dec 25;126(4):847–857. doi: 10.1016/0022-2836(78)90023-2. [DOI] [PubMed] [Google Scholar]
  8. Glickman B. W., Ripley L. S. Structural intermediates of deletion mutagenesis: a role for palindromic DNA. Proc Natl Acad Sci U S A. 1984 Jan;81(2):512–516. doi: 10.1073/pnas.81.2.512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Inselburg J. Replication of colicin E1 plasmid DNA in minicells from a unique replication initiation site. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2256–2259. doi: 10.1073/pnas.71.6.2256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jones I. M., Primrose S. B., Ehrlich S. D. Recombination between short direct repeats in a recA host. Mol Gen Genet. 1982;188(3):486–489. doi: 10.1007/BF00330053. [DOI] [PubMed] [Google Scholar]
  11. Lacatena R. M., Cesareni G. Interaction between RNA1 and the primer precursor in the regulation of Co1E1 replication. J Mol Biol. 1983 Nov 5;170(3):635–650. doi: 10.1016/s0022-2836(83)80125-9. [DOI] [PubMed] [Google Scholar]
  12. Lovett M. A., Katz L., Helinski D. R. Unidirectional replication of plasmid ColE1 DNA. Nature. 1974 Sep 27;251(5473):337–340. doi: 10.1038/251337a0. [DOI] [PubMed] [Google Scholar]
  13. Mandecki W. Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7177–7181. doi: 10.1073/pnas.83.19.7177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Marvo S. L., King S. R., Jaskunas S. R. Role of short regions of homology in intermolecular illegitimate recombination events. Proc Natl Acad Sci U S A. 1983 May;80(9):2452–2456. doi: 10.1073/pnas.80.9.2452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nag D. K., DasGupta U., Adelt G., Berg D. E. IS50-mediated inverse transposition: specificity and precision. Gene. 1985;34(1):17–26. doi: 10.1016/0378-1119(85)90290-2. [DOI] [PubMed] [Google Scholar]
  16. Noll M. Internal structure of the nucleosome: DNA folding in the conserved 140-base-pair core particle. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 1):77–85. doi: 10.1101/sqb.1978.042.01.009. [DOI] [PubMed] [Google Scholar]
  17. Schaaper R. M., Danforth B. N., Glickman B. W. Mechanisms of spontaneous mutagenesis: an analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene. J Mol Biol. 1986 May 20;189(2):273–284. doi: 10.1016/0022-2836(86)90509-7. [DOI] [PubMed] [Google Scholar]
  18. Singer B. S., Westlye J. Deletion formation in bacteriophage T4. J Mol Biol. 1988 Jul 20;202(2):233–243. doi: 10.1016/0022-2836(88)90454-8. [DOI] [PubMed] [Google Scholar]
  19. Streisinger G., Okada Y., Emrich J., Newton J., Tsugita A., Terzaghi E., Inouye M. Frameshift mutations and the genetic code. This paper is dedicated to Professor Theodosius Dobzhansky on the occasion of his 66th birthday. Cold Spring Harb Symp Quant Biol. 1966;31:77–84. doi: 10.1101/sqb.1966.031.01.014. [DOI] [PubMed] [Google Scholar]
  20. Tomizawa J. I., Ohmori H., Bird R. E. Origin of replication of colicin E1 plasmid DNA. Proc Natl Acad Sci U S A. 1977 May;74(5):1865–1869. doi: 10.1073/pnas.74.5.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Weston-Hafer K., Berg D. E. Limits to the role of palindromy in deletion formation. J Bacteriol. 1991 Jan;173(1):315–318. doi: 10.1128/jb.173.1.315-318.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weston-Hafer K., Berg D. E. Specificity of deletion events in pBR322. Plasmid. 1989 May;21(3):251–253. doi: 10.1016/0147-619x(89)90050-4. [DOI] [PubMed] [Google Scholar]

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