Reorganization of the Mu transpososome active sites during a cooperative transition between DNA cleavage and joining.
Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues. Mu transposase repeatedly utilizes the same active site DDE residues to cleave and join a single DNA strand at each transposon end to a new, distant DNA location (the target DNA). To better understand how DNA is manipulated within the Mu transposase-DNA complex during recombination, the impact of the DNA immediately adjacent to the Mu DNA ends (the flanking DNA) on the progress of transposition was investigated. We show that, in the absence of the MuB activator, the 3 '-flanking strand can slow one or more steps between DNA cleavage and joining. The presence of this flanking DNA strand in just one active site slows the joining step in both active sites. Further evidence suggests that this slow step is not due to a change in the affinity of the transpososome for the target DNA. Finally, we demonstrate that MuB activates transposition by stimulating the reaction step between cleavage and joining that is otherwise slowed by this flanking DNA strand. Based on these results, we propose that the 3 '-flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Tanya L
TL
Williams
Tania A
TA
Baker
D010583:Bacteriophage mu / Q000378:metabolism*; D010584:Bacteriophage phi X 174 / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D004251:DNA Transposable Elements / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008954:Models, Biological; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D011995:Recombination, Genetic; D013997:Time Factors
D004251:DNA Transposable Elements; D009841:Oligonucleotides; D004247:DNA; D003850:Deoxyribonuclease I