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K Kirkegaard

Publications and source records attributed to K Kirkegaard.

43 records · Page 3Linked to original sources
PubMed1985

Changes in the DNA structure of the lac UV5 promoter during formation of an open complex with Escherichia coli RNA polymerase.

By chemical and enzymatic methods, two stable complexes between Escherichia coli RNA polymerase and a linear DNA fragment carrying the lac UV5 promoter have been identified. In these binary complexes, DNA can adopt two alternate conformations as a function of temperature. Contacts between RNA polymerase and the DNA phosphate backbone are indistinguishable in these two forms, as revealed by probing with pancreatic DNase I. Protection of enhancement of the reactivity of the bases toward (CH3)2SO4 occurs, however, only in the form that predominates above 22 degrees C, RPo. The form stable at low temperature, RPi, is a "closed" complex since no single-stranded region is detectable in the DNA. The strong temperature dependence of the equilibrium constant, the midpoint value of the transition, and the rate of conversion between these two forms are in close agreement with a series of measurements performed by using a transcriptional assay and reported in the preceding paper [Buc, H., & McClure, W. R. (1985) Biochemistry (preceding paper in this issue)]. These data further support the postulated mechanism of open complex formation involving three sequential steps: R + P in equilibrium RPc in equilibrium RPi in equilibrium RPo. The binary complex RPc, which accumulates transiently at 37 degrees C before the isomerization leading to open complex formation, is not significantly protected against enzymatic cleavage or chemical modification and is therefore distinct from RPi and RPo.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1983

Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes.

A method based on the differential rate of cytosine methylation in single- and double-stranded nucleic acids by dimethyl sulfate [Peattie, D.A. & Gilbert, W. (1980) Proc. Natl. Acad. Sci. USA 77, 4679-4682] has been developed for probing unpaired cytosines in DNA and DNA-protein complexes at the sequence level. Application of the method to the complexes between Escherichia coli RNA polymerase (EC 2.7.7.6) and three related promoters, lac UV5, trp, and a hybrid promoter tac resulting from the fusion of the two, reveals distinct differences in the way RNA polymerase unpairs DNA in these promoters. No single-stranded region is detectable in the complex with the trp promoter. For the lac UV5 promoter, the cytosines at positions -6, -4, -2, and -1 are in an unpaired region. The same cytosines in the tac promoter, which is homologous in sequence to lac UV5 in this region, are also found to be single stranded. For the pair of promoters lac UV5 and tac, the cytosine methylation reaction has also been used to demonstrate the steep temperature dependence of opening of base pairs by RNA polymerase. One striking feature is that the midpoint of this transition for the tac promoter is 3 degrees C lower than the corresponding value for lac UV5, even though the sequence of the unpaired region in the two promoters is identical.

Base Sequence↗
PubMed1981

Mapping the topography of DNA wrapped around gyrase by nucleolytic and chemical probing of complexes of unique DNA sequences.

Complexes between DNA gyrase and DNA fragments of unique sequences were used to probe the topography of the DNA with nucleases and dimethyl sulfate. The results indicate that the flanking regions, each 50 bp in size, of a 145--155 bp DNA segment resistant to staphylococcal nuclease contain groups of pancreatic DNAase I-susceptible sites that are spaced 10--11 nucleotides apart. Pairs of adjacent DNAase I-sensitive sites on complementary strands are typically staggered by 2--4 bp. The binding of DNA to gyrase confers no protection against alkylation of the DNA by dimethyl sulfate. These properties of the gyrase-DNA complex are reminiscent of those of the nucleosome, and the common underlying structural feature appears to be wrapping of the DNA around a protein core. The gyrase-DNA complex differs from the nucleosome, however, in that it must possess features necessary for the catalysis of DNA chain breakage and the modulation of the DNA-enzyme interaction by ATP. We present evidence that the breakage and rejoining of the DNA by gyrase occur within a central region of the staphylococcal nuclease-resistant DNA segment. The relation of this observation to the mechanism of DNA supercoiling by gyrase is discussed. Addition of ATP or its beta, gamma-imido analog has essentially no effect on the patterns of susceptibilities to DNAase I, implying that the DNA-enzyme contacts mapped by the nuclease ae little affected by ATP-induced conformational changes.

Adenosine Triphosphate↗
PubMed1978

Escherichia coli DNA topoisomerase I catalyzed linking of single-stranded rings of complementary base sequences.

Eco DNA topoisomerase I (E. coli omega protein) has been observed to catalyze the formation of double-stranded, covalently closed DNA from complementary single-stranded DNA rings, a novel reaction which is topologically forbidden without the enzyme-catalyzed breakage and rejoining of DNA backbone bonds. Incubation of a mixture of single-stranded PM2 DNA rings of complementary base sequences with omega yields a species with a sedimentation coefficient in an alkaline medium characteristic of a covalently closed circular double-stranded DNA. Buoyant density measurements in CsCl at alkaline pH also identify the product as a covalently closed duplex ring. If the omega-catalyzed reaction is stopped short of completion, highly negatively supercoiled molecules are formed which sediment more slowly in an alkaline medium than the final duplex product. As the reaction proceeds the mean sedimentation rate of the intermediates increases. This is in agreement with the expectation that the linking number between the two complementary rings increases gradually during the course of the reaction from zero to that of a relaxed covalently closed circular DNA duplex. The possible role of DNA topoisomerases in genetic recombination is discussed.

Base Sequence↗