DNA-protein cooperativity in the assembly and stabilization of mu strand transfer complex. Relevance of DNA phasing and att site cleavage.
The requirements for negatively supercoiled DNA substrates, the cis-acting transposition enhancer and the Escherichia coli HU protein during the phage Mu transposition reaction are relaxed under DMSO-assay conditions. We have used these modified assay conditions to extend studies on the transposition pathway. We show here that linear DNA fragments containing the right end of Mu (attR) and Mu A protein mutually promote the assembly of "high-order" complexes held together by non-covalent protein-DNA and protein-protein interactions. A large subset of these complexes is competent in mediating strand transfer. DNA fragments containing the left end of Mu (attL) as well as non-Mu DNA can be used as targets during strand transfer. The R1 and R2 subsites within attR are required, but R3 is dispensable, in the protein-DNA oligomerization steps as well as in the strand transfer reaction. Proper phasing and spacing between R1 and R2 are central to the reaction. A single base-pair change in the terminal nucleotide that renders attR non-cleavable prevents the assembly of stable high-order complexes, showing that strand cleavage and stabilization of high-order complexes are tightly coupled events. Conversely, pre-cleavage at the attL site allows it to function in the assembly process, albeit at a much lower efficiency than attR. In the presence of HU, the reactivity of pre-cleaved attL is enhanced significantly.
Namgoong
S Y
SY
Department of Microbiology, University of Texas at Austin 78712.
Jayaram
M
M
Kim
K
K
Harshey
R M
RM
D001287:Attachment Sites, Microbiological; D001426:Bacterial Proteins / Q000378:metabolism; D010583:Bacteriophage mu / Q000235:genetics* / Q000378:metabolism; D001681:Biological Assay / Q000379:methods; D004251:DNA Transposable Elements / Q000502:physiology*; D004279:DNA, Viral / Q000032:analysis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004121:Dimethyl Sulfoxide; D004926:Escherichia coli / Q000737:chemistry; D008957:Models, Genetic; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D019895:Transposases
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C040330:histone-like protein HU, bacteria; D009713:Nucleotidyltransferases; D019895:Transposases; D004121:Dimethyl Sulfoxide