Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes.

Stable protein-DNA complexes or transpososomes mediate the Mu DNA strand transfer reaction in vitro (Surette, M. G., Buch, S. J., and Chaconas, G. (1987) Cell 49, 253-262; Craigie, R., and Mizuuchi, K. (1987) Cell 51, 493-501). Formation of the Type 1 complex, an intermediate in the strand transfer reaction, requires the Mu A and Escherichia coli HU proteins. Generation of the Type 2 complex, in which the Mu ends have been covalently linked to the target DNA, requires the Mu B protein, ATP, and target DNA in addition to A and HU. The protein content of these higher order synaptic complexes has been studied by immunoelectron microscopy using protein A-colloidal gold conjugates to visualize antibody-bound complexes. Under our in vitro transposition conditions, Type 1 complexes were found to contain A and HU; in addition, Type 2 complexes contained Mu B. However, both the HU and the Mu B protein were found to be loosely associated and could be quantitatively removed from the nucleoprotein core of both complexes by incubation in 0.5 M NaCl. Depletion of HU from the Type 1 complex did not affect the ability of this complex to be converted into the strand-transferred product. Hence, the indispensable role of the HU protein in the Mu DNA strand transfer reaction is limited to the formation of the Type 1 transpososome.

Determination of the secondary structure of the DNA binding protein Ner from phage Mu using 1H homonuclear and 15N-1H heteronuclear NMR spectroscopy.

The sequential resonance assignment of the 1H and 15N NMR spectra of the DNA binding protein Ner from phage Mu is presented. This is carried out by using a combination of 1H-1H and 1H-15N two-dimensional experiments. The availability of completely labeled 15N protein enabled us to record a variety of relayed heteronuclear multiple quantum coherence experiments, thereby enabling the correlation of proton-proton through-space and through-bond connectivities with the chemical shift of the directly bonded 15N atom. These heteronuclear experiments were crucial for the sequential assignment as the proton chemical shift dispersion of the Ner protein is limited and substantial overlap precluded unambiguous assignment of the homonuclear spectra in several cases. From a qualitative interpretation of the NOE data involving the NH, C alpha H, and C beta H protons, it is shown that Ner is composed of five helices extending from residues 11 to 22, 27 to 34, 38 to 45, 50 to 60, and 63 to 73.

[Construction and characteristics of mini-Mu phages]. / Konstruirovanie i kharakteristika mini-Mu-fagov.

The paper reports on the principles of construction, physical characterization and results of preliminary genetic investigation of hybrid plasmids containing Mu DNA sequences or deletion derivatives of phage Mu, the so-called mini-Mu phages. The mini-Mu were obtained by joining both phage ends within one plasmid in a regular orientation. A collection obtained by in vitro manipulations included 14 recombinant plasmids containing different DNA fragments of the Mu genome. Seven plasmids have both ends of phage Mu, three plasmids containing regularly oriented ends, i.e. mini-phages of different size: the mini-Mu5 (11 kb) within pRM8 plasmid, the mini-Mu4 Ap (18 kb) within pRM6 and the mini-mini-Mu (4.4 kb) within pRM5. The collection comprises mini-Mu phages with the gene kil inactivated after treatment with hydroxylamine. Biological properties of the hybrid plasmids have been preliminary studied.

Partial purification and properties of an exonuclease inhibitor induced by bacteriophage Mu-1.

From an induced lysogen of bacteriophage Mu-1, we partially purified a substance of high molecular weight that blocks the action of several exonucleases on double-stranded DNA. The presence of the inhibitor in cell-free extracts is dependent on induction of a Mu prophage. The Mu-related inhibitor acts by binding to double-stranded DNA rather than by interacting with the DNase. The inhibitor protects linear duplex DNA of Mu, P22, and phi X174am3 from exonucleolytic degradation by recBC DNase and lambda exonuclease. Single-stranded DNA, however, is not protected by the inhibitor from degradation by either recBC DNase or exonuclease I. The inhibitor preparation contains a protein that binds to linear duplex DNA, but not to circular duplex DNA; ends are required for binding to occur. Single-stranded DNA is not a substrate for the binding protein. These and other results suggest that the binding protein and the inhibitor are the same activity.