Skip to main content
Genetics logoLink to Genetics
. 1994 Jun;137(2):343–352. doi: 10.1093/genetics/137.2.343

Intragenic Suppression of Integration-Defective Is10 Transposase Mutants

M S Junop 1, D Hockman 1, D B Haniford 1
PMCID: PMC1205960  PMID: 8070648

Abstract

IS10 transposase mediates excision and integration reactions in Tn10/IS10 transposition. Mutations in IS10 transposase that specifically block integration have previously been identified; however, the mechanism by which these mutations block integration has not been established. One approach to defining the basis of this block is to identify ways in which the original defect can be corrected. The approach we have taken toward this end has been to isolate and characterize intragenic second site suppressors to two different integration-defective mutants. Of the second site suppressors identified, one, CY134, is of particular interest for two reasons. First, it suppresses at least seven different mutations that confer an integration-defective phenotype. Interestingly, these mutations map in two separate segments of transposase, designated patch I and patch II. Second, CY134 on its own has previously been shown to relax the target DNA sequence requirements for Tn10 integration. We provide evidence that suppression by CY134 is not simply a consequence of this mutation conferring a general ``transposition up'' phenotype, but rather is due to correcting the original defect. Possible mechanisms of suppression for both CY134 and other second site suppressors are considered.

Full Text

The Full Text of this article is available as a PDF (6.3 MB).

Selected References

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

  1. Bender J., Kleckner N. IS10 transposase mutations that specifically alter target site recognition. EMBO J. 1992 Feb;11(2):741–750. doi: 10.1002/j.1460-2075.1992.tb05107.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benjamin H. W., Kleckner N. Intramolecular transposition by Tn10. Cell. 1989 Oct 20;59(2):373–383. doi: 10.1016/0092-8674(89)90298-5. [DOI] [PubMed] [Google Scholar]
  3. Clewell D. B., Helinski D. R. Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry. 1970 Oct 27;9(22):4428–4440. doi: 10.1021/bi00824a026. [DOI] [PubMed] [Google Scholar]
  4. Ermácora M. R., Delfino J. M., Cuenoud B., Schepartz A., Fox R. O. Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6383–6387. doi: 10.1073/pnas.89.14.6383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Haniford D. B., Benjamin H. W., Kleckner N. Kinetic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tn10 transposition. Cell. 1991 Jan 11;64(1):171–179. doi: 10.1016/0092-8674(91)90218-n. [DOI] [PubMed] [Google Scholar]
  6. Haniford D. B., Chaconas G. Mechanistic aspects of DNA transposition. Curr Opin Genet Dev. 1992 Oct;2(5):698–704. doi: 10.1016/s0959-437x(05)80129-7. [DOI] [PubMed] [Google Scholar]
  7. Haniford D. B., Chelouche A. R., Kleckner N. A specific class of IS10 transposase mutants are blocked for target site interactions and promote formation of an excised transposon fragment. Cell. 1989 Oct 20;59(2):385–394. doi: 10.1016/0092-8674(89)90299-7. [DOI] [PubMed] [Google Scholar]
  8. Hecht M. H., Sauer R. T. Phage lambda repressor revertants. Amino acid substitutions that restore activity to mutant proteins. J Mol Biol. 1985 Nov 5;186(1):53–63. doi: 10.1016/0022-2836(85)90256-6. [DOI] [PubMed] [Google Scholar]
  9. Huisman O., Kleckner N. A new generalizable test for detection of mutations affecting Tn10 transposition. Genetics. 1987 Jun;116(2):185–189. doi: 10.1093/genetics/116.2.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mahillon J., Seurinck J., van Rompuy L., Delcour J., Zabeau M. Nucleotide sequence and structural organization of an insertion sequence element (IS231) from Bacillus thuringiensis strain berliner 1715. EMBO J. 1985 Dec 30;4(13B):3895–3899. doi: 10.1002/j.1460-2075.1985.tb04163.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nelson D. R., Douglas M. G. Function-based mapping of the yeast mitochondrial ADP/ATP translocator by selection for second site revertants. J Mol Biol. 1993 Apr 20;230(4):1171–1182. doi: 10.1006/jmbi.1993.1234. [DOI] [PubMed] [Google Scholar]
  12. Poteete A. R., Sun D. P., Nicholson H., Matthews B. W. Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft. Biochemistry. 1991 Feb 5;30(5):1425–1432. doi: 10.1021/bi00219a037. [DOI] [PubMed] [Google Scholar]
  13. Shortle D., Lin B. Genetic analysis of staphylococcal nuclease: identification of three intragenic "global" suppressors of nuclease-minus mutations. Genetics. 1985 Aug;110(4):539–555. doi: 10.1093/genetics/110.4.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Singer M., Jin D. J., Walter W. A., Gross C. A. Genetic evidence for the interaction between cluster I and cluster III rifampicin resistant mutations. J Mol Biol. 1993 May 5;231(1):1–5. doi: 10.1006/jmbi.1993.1251. [DOI] [PubMed] [Google Scholar]
  15. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES