Linkage map of Escherichia coli K-12, edition 8.

A wide range of cytotoxic and mutagenic DNA bases are removed by different DNA glycosylases, which initiate the base excision repair pathway. DNA glycosylases cleave the N-glycosylic bond between the target base and deoxyribose, thus releasing a free base and leaving an apurinic/apyrimidinic (AP) site. In addition, several DNA glycosylases are bifunctional, since they also display a lyase activity that cleaves the phosphodiester backbone 3' to the AP site generated by the glycosylase activity. Structural data and sequence comparisons have identified common features among many of the DNA glycosylases. Their active sites have a structure that can only bind extrahelical target bases, as observed in the crystal structure of human uracil-DNA glycosylase in a complex with double-stranded DNA. Nucleotide flipping is apparently actively facilitated by the enzyme. With bacteriophage T4 endonuclease V, a pyrimidine-dimer glycosylase, the enzyme gains access to the target base by flipping out an adenine opposite to the dimer. A conserved helix-hairpin-helix motif and an invariant Asp residue are found in the active sites of more than 20 monofunctional and bifunctional DNA glycosylases. In bifunctional DNA glycosylases, the conserved Asp is thought to deprotonate a conserved Lys, forming an amine nucleophile. The nucleophile forms a covalent intermediate (Schiff base) with the deoxyribose anomeric carbon and expels the base. Deoxyribose subsequently undergoes several transformations, resulting in strand cleavage and regeneration of the free enzyme. The catalytic mechanism of monofunctional glycosylases does not involve covalent intermediates. Instead the conserved Asp residue may activate a water molecule which acts as the attacking nucleophile.

DNA glycosylases in the base excision repair of DNA.

Novel oligomers are disclosed which have enhanced ability with respect to forming duplexes or triplexes compared with oligomers containing only conventional bases. The oligomers contain the bases 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine or related analogs. The oligomers of the invention are capable of (i) forming triplexes with various target sequences such as virus or oncogene sequences by coupling into the major groove of a target DNA duplex at physiological pH or (ii) forming duplexes by binding to single-stranded DNA or to RNA encoded by target genes. The oligomers of the invention can be incorporated into pharmaceutically acceptable carriers and can be constructed to have any desired sequence, provided the sequence normally includes one or more bases that is replaced with the analogs of the invention. Compositions of the invention can be used as pharmaceutical agents to treat various diseases such as those caused by viruses and can be used for diagnostic purposes in order to detect viruses or disease conditions.

Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines

https://mmbr.asm.org/content/mmbr/51/3/301.full.pdf

Histonelike proteins of bacteria.

In order to localize the cells expressing 5-HT1A receptors in the rat brain, we used in situ hybridization histochemistry to visualize the distribution of the mRNA coding for 5-HT1A receptors. Oligonucleotides derived from different parts of the coding region of the rat 5-HT1A receptor gene were used as hybridization probes. 5-HT1A binding sites were visualized on consecutive sections by receptor autoradiography using 3H-8-hydroxy-2-(di-n-propylamino)tetralin as ligand. The highest levels of hybridization were observed in the dorsal raphe nucleus, septum, hippocampus, entorhinal cortex, and interpeduncular nucleus. Positive hybridization signals were also present in other areas, such as the olfactory bulb; cerebral cortex; some thalamic and hypothalamic nuclei; several nuclei of the brainstem, including all the remaining raphe nuclei, nucleus of the solitary tract, and nucleus of the spinal tract of the trigeminus; and the dorsal horn of the spinal cord. The distribution and abundance of 5-HT1A receptor mRNA in different rat brain areas generally correlate with those of the binding sites, suggesting that 5-HT1A receptors are predominantly somatodendritic receptors.

https://www.jneurosci.org/content/jneuro/12/2/440.full.pdf

Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding

Applications of magnetic beads with covalently attached oligonucleotides in hybridization: Isolation and detection of specific measles virus mRNA from a crude cell lysate

Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes.

Polyamine induced aggregation of various DNAs has been studied under conditions usually employed in many enzymatic assays where DNA is one of the substrates. Spermine was by far the most efficient polyamine in causing aggregation followed by spermidine and cadaverine. All double-stranded and naturally occurring single-stranded DNAs were found to aggregate. No aggregation of single-stranded homodeoxypolymers could be detected under the same conditions. The concentration of polyamine at which the aggregation commenced was found to be a linear function of the DNA concentration. The slope of the curves depended on the nature of the polyamine, DNA the concentration of Mg++ and the ionic strength.

Polyamine induced aggregation of DNA

This article deals with some aspects of the structure of the nucleoid in Escherichia coli. It is based on a contribution to the symposium on' Relations between Structure and Function in the Prokaryotic Cell' at the Annual Meeting of the Society for General Microbiology in April 1978, but was not published in the Symposium Volume.

/pdf/the-bacterial-nucleoid-55qtcxw8qo.pdf

The Bacterial Nucleoid

The kinetics of T4 polynucleotide ligase has been investigated at pH 8, 20 °C and using the double-stranded DNA substrate (dA)n [(dT)10]n/10. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true Km for the DNA substrate was 0.6 μM and that of ATP 100 μM.



Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32P-labelled (dA)n [(dT)40]n/40 as substrate. No breakdown of this DNA could be detected.



The joining reaction was inhibited by high concentrations, i.e. above approximately 70 mM, of salts such as KC1, NaCl, NH4Cl and CsC1. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1975.tb21021.x

Kjell Kleppe

Papers

Applications of magnetic beads with covalently attached oligonucleotides in hybridization: Isolation and detection of specific measles virus mRNA from a crude cell lysate

Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes.

Polyamine induced aggregation of DNA

The Bacterial Nucleoid

Kinetics and Effect of Salts and Polyamines on T4 Polynucleotide Ligase