Abstract
During Tn10 transposition, the element is excised from the donor site by double-strand cleavages at the two transposon ends. Double-strand cleavage is a central step in the nonreplicative transposition reaction of many transposons in both prokaryotes and eukaryotes. Evidence is presented to show that the Tn10 double-strand cut is made by an ordered, sequential cleavage of the two strands. The transferred strand is cut first, and then the nontransferred strand is cleaved. The single-strand nicked intermediate is seen to accumulate when Mn2+ is substituted for Mg2+ in the reaction or when certain mutant transposases are used. The fact that the transferred strand is cleaved before the non-transferred strand implies that the order of strand cleavages is not the determining factor that precludes a replicative mechanism of transposition.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bainton R. J., Kubo K. M., Feng J. N., Craig N. L. Tn7 transposition: target DNA recognition is mediated by multiple Tn7-encoded proteins in a purified in vitro system. Cell. 1993 Mar 26;72(6):931–943. doi: 10.1016/0092-8674(93)90581-a. [DOI] [PubMed] [Google Scholar]
- Bender J., Kleckner N. Genetic evidence that Tn10 transposes by a nonreplicative mechanism. Cell. 1986 Jun 20;45(6):801–815. doi: 10.1016/0092-8674(86)90555-6. [DOI] [PubMed] [Google Scholar]
- Chalmers R. M., Kleckner N. Tn10/IS10 transposase purification, activation, and in vitro reaction. J Biol Chem. 1994 Mar 18;269(11):8029–8035. [PubMed] [Google Scholar]
- 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]
- 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]
- Haniford D., Kleckner N. Tn 10 transposition in vivo: temporal separation of cleavages at the two transposon ends and roles of terminal basepairs subsequent to interaction of ends. EMBO J. 1994 Jul 15;13(14):3401–3411. doi: 10.1002/j.1460-2075.1994.tb06643.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huisman O., Errada P. R., Signon L., Kleckner N. Mutational analysis of IS10's outside end. EMBO J. 1989 Jul;8(7):2101–2109. doi: 10.1002/j.1460-2075.1989.tb03619.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Mizuuchi K., Craigie R. Mechanism of bacteriophage mu transposition. Annu Rev Genet. 1986;20:385–429. doi: 10.1146/annurev.ge.20.120186.002125. [DOI] [PubMed] [Google Scholar]
- Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]