Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification

The alternate expression of the Salmonella flagellin genes H1 and H2 is controlled by the orientation of a 995-base-pair invertible segment of DNA located at the 5' end of the H2 gene. The hin gene, which is encoded within the invertible region, is essential for the inversion of this DNA segment. We cloned the hin gene into Escherichia coli and placed it under the control of the PL promoter of bacteriophage lambda. These cells overproduced the Hin protein. In vivo inversion activity was measured by using a recombinant lambda phage which contains the H2 and lacZ genes under the control of the invertible region. Using this phage, we showed that the amount of inversion activity is proportional to the amount of Hin protein in the cell. An inactive form of the protein was purified by using the unusual solubility properties of the overproduced protein. The amino acid composition of the protein agreed with the DNA sequence of the hin gene. Antibodies were made to the isolated protein. These antibodies cross-reacted with two other unidentified E. coli proteins

Why a Particle Physicist is Interested in DNA Branch Migration

We describe an explicitly discrete model of the process of DNA branch migration. The model matches the existing data well, but we find that branch migration along long strands of DNA (N \simge 40~bp) is also well modeled by continuum diffusion. The discrete model is still useful for guiding future experiments.Comment: Talk presented at LATTICE96(theoretical developments); 3 pages, TeXsis w/ LAT96.txs (available from ftp://lifshitz.ph.utexas.edu/texsis/styles/LAT96.txs and will be a part of the next Elsevier.txs) and TXSdcol.te

Secondary structural characterization of oligonucleotide strands using electrospray ionization mass spectrometry

Differences in charge state distributions of hairpin versus linear strands of oligonucleotides are analyzed using electrospray ionization mass spectrometry (ESI-MS) in the negative ion detection mode. It is observed that the linear structures show lower charge state distribution than the hairpin strands of the same composition. The concentration of ammonium acetate and the cone voltage are major factors that cause the shift of the negative ions in the charge states. The ESI data presented here are supported by UV spectra of strands acquired at 260 nm wavelength in aqueous ammonium acetate solution. We will show that the strands that demonstrate a higher charge state distribution in the gas phase also have a higher melting temperature in solution

DNA-binding properties of the Hin recombinase

The recombinase of the Salmonella inversion system, Hin, mediates site-specific recombination between two 26 base pairs (bp) inverted repeat sequences (hixL and hixR) which flank a 993-bp DNA segment. We have investigated Hin recognition of, and association with, the hix recombination sites. Nuclease and chemical protection studies with linear and supercoiled DNA substrates demonstrate that Hin initially binds hixL and hixR independently of binding of the other protein components of the inversion system, Fis and HU. DNA-binding assays with mutant recombination sites and methylation interference experiments indicate that the critical bases for Hin recognition of its DNA-binding site are within an 8-bp sequence covering adjacent major and minor grooves of the DNA helix in each of the 12-bp half-sites of the hix recombination sites. The nature of the Hin-hix complexes in these binding studies and the results of gel filtration assays with purified Hin suggests that Hin binds the recombination sites as a dimer. The implications of the nature of the interactions of Hin with its recombination sites on the mechanism of the recombination reaction and on the novel features of DNA recognition by Hin are discussed

Synthesis of a sequence-specific DNA-cleaving peptide

A synthetic 52-residue peptide based on the sequence-specific DNA-binding domain of Hin recombinase (139-190) has been equipped with ethylenediaminetetraacetic acid (EDTA) at the amino terminus. In the presence of Fe(II), this synthetic EDTA-peptide cleaves DNA at Hin recombination sites. The cleavage data reveal that the amino terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin recombination sites. This work demonstrates the construction of a hybrid peptide combining two functional domains: sequence-specific DNA binding and DNA cleavage

Synthesis of a Site-Specific DNA-Binding Peptide

The Hin recombinase binds to specific sites on DNA and mediates a recombination event that results in DNA inversion. In order to define the DNA-binding domain of the Hin protein two peptides 31 and 52 amino acids long were synthesized. Even though the 31mer encompassed the sequence encoding the putative helix-coil-helix-binding domain, it was not sufficient for binding to the 26-base pair DNA crossover site. However, the 52mer specifically interacted with the site and also effectively inhibited the Hin-mediated recombination reaction. The 52mer bound effectively to both the 26-base pair complete site and to a 14-base pair "half site." Nuclease and chemical protection studies with the 52mer helped to define the DNA base pairs that contributed to the specificity of binding. The synthetic peptide provides opportunities for new approaches to the study of the nature of protein-DNA interaction