Abstract
We have examined the supercoiling requirement for the in vitro Mu DNA strand transfer reaction and found that optimal efficiency requires a high level (sigma = -0.06) of donor plasmid superhelicity. At in vivo levels of supercoiling (sigma = -0.025) the reaction does not occur. Using an unreactive donor plasmid with a near physiological level of supercoiling, we identified an Escherichia coli protein factor which has the novel property of reducing the donor plasmid supercoiling requirement for the in vitro Mu DNA strand transfer reaction by 40%. This protein, which we named supercoiling relief factor was purified to near homogeneity and found to be identical to integration host factor (IHF), a protein known to induce site specific bends in DNA. The dramatic reduction in the supercoiling requirement was promoted by about 1.5 IHF dimers/donor substrate molecule. At these low levels of IHF, the HU requirement for the reaction was also reduced; a synergistic effect of the two proteins resulted in a greater than 10-fold stimulation of the reaction under appropriate conditions. Furthermore, at high concentrations of IHF, HU could be completely eliminated from the reaction.
Highlights
A Protein Factor Which Reduces theNegative Supercoiling Requirement in the Mu DNA Strand Transfer ReactionIs Escherichia coli Integration Host Factor*
Using an unreactive donor plasmid with a near physiological level of supercoiling, we identified an Escherichiacoli protein factor which has the novel property of reducing the donor plasmid supercoiling requirement for the in vitro Mu DNA strand transfer reaction by 40%.This protein, which we named supercoiling relief factor was purified to near homogeneity andfound to be identical to integration host factor (IHF),a protein known to induce site specific bends in DNA
Formation of the Tn3 resolvase synaptic complex is predicted to be favored by negative supercoiling; in this case negative plectonemic supercoils are believed to be constrained in the complex (Benjamin and Cozzarelli, 1988).A second way in which DNA
Summary
XhoI and PuuII andcloned into thelarge fragment of pBR322 which had been digested with Sal andPuuIIto generate pGG215. Because our HU preparations were usually stored in Hepes buffer, which is unsuitable for absorbance measurements at 205 nm, we routinely determined HU concentrations by trichloroacetic acidLowry assays (Peterson, 1977)using bovine serum albumin standards; the HU concentrations from the Lowry assays were multiplied by a correction factor of1.25 to bring them into agreement with values obtained from absorbance at 205 nm. Under our reaction conditions of 140 mM NaCl no measurable wrapping of HU was observed as judged by the lack of retention of negative supercoils upon relaxation of the DNA with wheat germ topoisomerase in the presence versus the absence of HU (datanot shown). The SRF activity eluted between 0.7-0.9 M NaCl. The concentration of SRF (and IHF) was determined using the trichloroacetic acid-Lowry method and bovine serum albumin as a standard (Peterson, 1977). Seven days after the last boost the rabbit was killed by heart puncture and theserum collected
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