Transposition of the human Hsmar1 transposon: rate-limiting steps and the importance of the flanking TA dinucleotide in second strand cleavage.
Hsmar1 is a member of the mariner family of DNA transposons. Although widespread in nature, their molecular mechanism remains obscure. Many other cut-and-paste elements use a hairpin intermediate to cleave the two strands of DNA at each transposon end. However, this intermediate is absent in mariner, suggesting that these elements use a fundamentally different mechanism for second-strand cleavage. We have taken advantage of the faithful and efficient in vitro reaction provided by Hsmar1 to characterize the products and intermediates of transposition. We report different factors that particularly affect the reaction, which are the reaction pH and the transposase concentration. Kinetic analysis revealed that first-strand nicking and integration are rapid. The rate of the reaction is limited in part by the divalent metal ion-dependent assembly of a complex between transposase and the transposon end(s) prior to the first catalytic step. Second-strand cleavage is the rate-limiting catalytic step of the reaction. We discuss our data in light of a model for the two metal ion catalytic mechanism and propose that mariner excision involves a significant conformational change between first- and second-strand cleavage at each transposon end. Furthermore, this conformational change requires specific contacts between transposase and the flanking TA dinucleotide.
School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, UK.
Corentin
C
Claeys Bouuaert
Ronald
R
Chalmers
D000227:Adenine Nucleotides / Q000737:chemistry; D053837:DNA Cleavage; D004251:DNA Transposable Elements; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D013942:Thymine Nucleotides / Q000737:chemistry; D019895:Transposases / Q000302:isolation & purification / Q000378:metabolism*
D000227:Adenine Nucleotides; D004251:DNA Transposable Elements; D008670:Metals; D013942:Thymine Nucleotides; D019895:Transposases