Analysis of P element transposase protein-DNA interactions during the early stages of transposition.
P elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.
Tang
Mei
M
Department of Molecular and Cell Biology, Division of Genetics, Genomics and Development and Division of Biochemistry and Molecular Biology, Center for Integrative Genomics, University of California, Berkeley, California 94720.
Cecconi
Ciro
C
CNR-INFM-S3 University of Modena e Reggio Emilia, 41100 Modena, Italy.
Bustamante
Carlos
C
Department of Molecular and Cell Biology, Division of Genetics, Genomics and Development and Division of Biochemistry and Molecular Biology, Center for Integrative Genomics, University of California, Berkeley, California 94720; Department of Physics, University of California, Berkeley, California 94720; Howard Hughes Medical Institute, Berkeley, California 94720; Lawrence Berkeley National Laboratory, Berkeley, California 94720.
Rio
Donald C
DC
Department of Molecular and Cell Biology, Division of Genetics, Genomics and Development and Division of Biochemistry and Molecular Biology, Center for Integrative Genomics, University of California, Berkeley, California 94720. Electronic address: don_rio@berkeley.edu.
D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D003067:Coenzymes / Q000737:chemistry* / Q000494:pharmacology; D004251:DNA Transposable Elements / Q000502:physiology; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D004331:Drosophila melanogaster; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D018625:Microscopy, Atomic Force; D020836:Protein Structure, Quaternary; D019895:Transposases / Q000737:chemistry* / Q000378:metabolism
D003067:Coenzymes; D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D006160:Guanosine Triphosphate; D019895:Transposases