Abstract
The tnpR gene of transposon Tn3 encodes a site-specific recombination enzyme that acts at res, a DNA region adjacent to tnpR, to convert co-integrate intermediates of interreplicon transposition to the normal transposition end-products. We have used two complementary approaches to study the nature of the Tn3 recombination region, res. Firstly, the DNA-binding sites for tnpR protein were determined in DNase I protection experiments. These identified a 120-bp region between the tnpA and tnpR genes that can be subdivided into three separate protein-binding sites. Genetic dissection experiments indicate that few, if any, other sequences in addition to this 120-bp region are required for res function. Moreover, we have shown that the two directly repeated res regions within a molecule are unequal partners in the recombination reaction: a truncated res region, which is unable to recombine with a second identical res region, can recombine efficiently with an intact res region. This demonstration, along with the observation that tnpR/res recombination acts efficiently on directly repeated res regions within a molecule but inefficiently both on inverted res regions in the same molecule and in the fusion reaction between res regions in different molecules, leads us to propose that one-dimensional diffusion (tracking) of tnpR protein along DNA is used to locate an initial res region, and then to bring a second directly repeated res region into a position that allows recombination between the res regions.
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arthur A., Sherratt D. Dissection of the transposition process: a transposon-encoded site-specific recombination system. Mol Gen Genet. 1979 Oct 1;175(3):267–274. doi: 10.1007/BF00397226. [DOI] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
- Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chiang S. J., Clowes R. C. Recombination between two TnA transposon sequences oriented as inverse repeats is found less frequently than between direct repeats. Mol Gen Genet. 1982;185(1):169–175. doi: 10.1007/BF00333809. [DOI] [PubMed] [Google Scholar]
- Finnegan J., Sherratt D. Plasmid ColE1 conjugal mobility: the nature of bom, a region required in cis for transfer. Mol Gen Genet. 1982;185(2):344–351. doi: 10.1007/BF00330810. [DOI] [PubMed] [Google Scholar]
- Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grindley N. D., Lauth M. R., Wells R. G., Wityk R. J., Salvo J. J., Reed R. R. Transposon-mediated site-specific recombination: identification of three binding sites for resolvase at the res sites of gamma delta and Tn3. Cell. 1982 Aug;30(1):19–27. doi: 10.1016/0092-8674(82)90007-1. [DOI] [PubMed] [Google Scholar]
- Heffron F., Bedinger P., Champoux J. J., Falkow S. Deletions affecting the transposition of an antibiotic resistance gene. Proc Natl Acad Sci U S A. 1977 Feb;74(2):702–706. doi: 10.1073/pnas.74.2.702. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kitts P. A., Lamond A., Sherratt D. J. Inter-replicon transposition of Tn1/3 occurs in two sequential genetically separable steps. Nature. 1982 Feb 18;295(5850):626–628. doi: 10.1038/295626a0. [DOI] [PubMed] [Google Scholar]
- Kitts P., Symington L., Burke M., Reed R., Sherratt D. Transposon-specified site-specific recombination. Proc Natl Acad Sci U S A. 1982 Jan;79(1):46–50. doi: 10.1073/pnas.79.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kleckner N. Transposable elements in prokaryotes. Annu Rev Genet. 1981;15:341–404. doi: 10.1146/annurev.ge.15.120181.002013. [DOI] [PubMed] [Google Scholar]
- Kostriken R., Morita C., Heffron F. Transposon Tn3 encodes a site-specific recombination system: identification of essential sequences, genes, and actual site of recombination. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4041–4045. doi: 10.1073/pnas.78.7.4041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- Reed R. R., Grindley N. D. Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site. Cell. 1981 Sep;25(3):721–728. doi: 10.1016/0092-8674(81)90179-3. [DOI] [PubMed] [Google Scholar]
- Reed R. R. Resolution of cointegrates between transposons gamma delta and Tn3 defines the recombination site. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3428–3432. doi: 10.1073/pnas.78.6.3428. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reed R. R. Transposon-mediated site-specific recombination: a defined in vitro system. Cell. 1981 Sep;25(3):713–719. doi: 10.1016/0092-8674(81)90178-1. [DOI] [PubMed] [Google Scholar]
- Robinson M. K., Bennett P. M., Richmond M. H. Inhibition of TnA translocation by TnA. J Bacteriol. 1977 Jan;129(1):407–414. doi: 10.1128/jb.129.1.407-414.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sherratt D., Arthur A., Burke M. Transposon-specified, site-specific recombination systems. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):275–281. doi: 10.1101/sqb.1981.045.01.040. [DOI] [PubMed] [Google Scholar]
- Simon M., Zieg J., Silverman M., Mandel G., Doolittle R. Phase variation: evolution of a controlling element. Science. 1980 Sep 19;209(4463):1370–1374. doi: 10.1126/science.6251543. [DOI] [PubMed] [Google Scholar]
- Tacon W., Bhamra S., Sunar B., Sherratt D. Structure and function of plasmid ColK. Plasmid. 1981 Nov;6(3):358–359. doi: 10.1016/0147-619x(81)90044-5. [DOI] [PubMed] [Google Scholar]