Site-specific HU binding in the Mu transpososome: conversion of a sequence-independent DNA-binding protein into a chemical nuclease.
HU is a small, basic, sequence-independent DNA-binding protein capable of engineering DNA deformations required for the formation of higher order nucleoprotein structures. One such complex is the Mu Type 1 transpososome, where the ends of Mu are stably synapsed by a tetramer of Mu A and cleaved at their 3' ends. HU is believed to play a critical role in transpososome assembly, which requires the communication of the two Mu ends and the transpositional enhancer. Although footprinting studies have clearly defined the DNA regions bound by Mu A, no protection could be ascribed to the HU protein by DNAse I, MPE.Fe(II) or hydroxyl radical methods (Lavoie et al. 1991). To directly probe HU interactions with the transpososome DNA, we have coupled HU to a DNA cleavage reagent, iron-EDTA, and report here the first HU "footprint". HU-nuclease cleavage was detectable at specific sites within an 83-bp spacer DNA separating the left-end-most L1 site from its neighboring L2 site. This HU binding was specific since it could not be competed with 10-fold excess supercoiled DNA. We postulate that HU promotes the formation of a tight DNA bend or loop in this region which facilitates the communication of Mu A monomers during complex assembly. This method may prove generally useful for the localization of sequence-independent DNA-binding proteins on DNA and within higher oder nucleoprotein structures.
Lavoie
B D
BD
Department of Biochemistry, University of Western Ontario, London, Canada.
Chaconas
G
G
D001426:Bacterial Proteins / Q000378:metabolism*; D010583:Bacteriophage mu / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C040330:histone-like protein HU, bacteria; D004247:DNA; D015253:Deoxyribonucleases, Type I Site-Specific