Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor

Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples such as biopsies. TDP1 removes covalent 3'DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5'fluorophore and a 3'quencher brought in close proximity by the secondary structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only "signal amplification" the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1 activity in pure enzyme fractions as well as in crude cell extracts. In the present study we demonstrate the specificity of the biosensor, its ability to quantitatively detect up- or down-regulated TDP1 activity, and that it may be used for measuring and for analyzing the mechanism of TDP1 inhibition

Effects of DNA supercoiling on chromatin architecture

Disruptions in chromatin structure are necessary for the regulation of eukaryotic genomes, from remodelling of nucleosomes at the base pair level through to large-scale chromatin domains that are hundreds of kilobases in size. RNA polymerase is a powerful motor which, prevented from turning with the tight helical pitch of the DNA, generates over-wound DNA ahead of itself and under-wound DNA behind. Mounting evidence supports a central role for transcription-dependent DNA supercoiling in disrupting chromatin structure at all scales. This supercoiling changes the properties of the DNA helix in a manner that substantially alters the binding specificity of DNA binding proteins and complexes, including nucleosomes, polymerases, topoisomerases and transcription factors. For example, transient over-wound DNA destabilises nucleosome core particles ahead of a transcribing polymerase, whereas under-wound DNA facilitates pre-initiation complex formation, transcription factor binding and nucleosome core particle association behind the transcribing polymerase. Importantly, DNA supercoiling can also dissipate through DNA, even in a chromatinised context, to influence both local elements and large chromatin domains. We propose a model in which changes in unconstrained DNA supercoiling influences higher levels of chromatin organisation through the additive effects of DNA supercoiling on both DNA-protein and DNA-nucleosome interactions. This model links small-scale changes in DNA and chromatin to the higher-order fibre and large-scale chromatin structures, providing a mechanism relating gene regulation to chromatin architecture in vivo

Skeletal remains from St. Peder’s parish church in Randers

Skeletal Remains from St. Peder's parish church in Randers One of Denmarks 17 known Benedictine nunneries was excavated in Randers, Jutland in 1989 (Matthiesen 1989). Although it is not clear when the nunnery was established, it seems that the church, which was part of the nunnery, was built around 1100 A.D. The church, St. Peders, also served as parish church (fig. 1). Two graveyards were located: one south of the church presumably used by the nuns, and a second east of the church which was probably the parish cemetery (Mikkelsen 1990a, Mikkelsen 1990b). The following is a brief account of the preliminary results of the anthropological analyses of the skeletal remains from the parish cemetery excavations. A minimum of 530 individuals were excavated, originating from 252 well defined single graves, several probable mass graves and stray finds (Mikkelsen & Matthiesen 1990). The most frequent type of burial was interment without coffin. Coffin burials were rare, and only six stone lined graves were found. It is assumed that the individuals found in the coffins and stone-lined graves represent people of a higher social status. The skeletons The preservation and completeness of the individual skeletons varied. Most of the large long bones were present. Roughly, one third of the material was well preserved, one third preserved to an average degree, and one third poorly preserved. It is noteworthy that 95 individuals were retrieved with all teeth present (apart from teeth lost ante mortem), and this testifies to the care with which the material was recovered. Age and sex distributions So far only the skeletal material from the single graves has been processed, yielding the following age distribution: 2 fetuses; 60 infants (0 to 12/14 years); 22 adolescents (12/14 to 21 years); and 215 adults (above 21 years) (fig. 2). Only few individuals were found to have an age exceeding 40 years. Subadults make up one third of the total material. Probably the real proportion was higher, up to 50%, due to the high child mortality of the period. However, the delicate bones of infants are not preserved to the same degree as the larger and denser bones of adults. The determination of sex is only possible for the adults, and revealed a fairly balanced distribution. Paleopathology Ordinary and well known pathological conditions affecting bones were found, e.g., many healed or partly healed fractures; inflammations and arthritis. One case of possible tuberculosis was found (see below). Generally, the types and rates of paleopathological changes agree with the results from other Medieval excavations (Møller-Christensen 1958). Although the early Benedictine nunneries to some extent functioned as hospitals (Trabjerg 1993), nothing definite can be deduced from the skeletons in this respect. Dental analyses revealed a low frequency of caries (88 individuals had caries), which is in agreement with results from other contemporary skeletal material (Mellquist & Sandberg 1939; Brinch & Møller­Christensen 1949). There was a clear correlation between caries and periapical abcesses, with half of the individuals with caries having abcesses (fig. 3). The degree of dental wear was moderate (fig. 4). Anthropometry The stature was calculated to be approx. 174 cm. (5' 9») for males and 157 cm. (5' 2») for females, well in accordance with other contemporary finds (Bennike 1985). Several other measurements, especially cranial measurements were taken, and these have since formed part of a large study of cranial morphology of Danish skeletons from the Stone Age to present days (Hjalgrim 1992). Special graves Although the purpose of the anthropological analyses was to reconstruct the population of the period, a few single cases attracted special attention: Grave 13: The skeleton of a young, approx. 18 years old female was found (fig. 5) including the bones of two fetuses (twins, see figure 5) in the pelvic region. From the examination of the fetal bones it appeared that the woman died about 6 months into her pregnancy (fig. 6). The cause of death could not be established. Stray find 510: A male cranium with the facial skeleton completely destroyed. A large iron ball, approx. 30 mm in diameter was found within the calvarium. Analyses at the Institute of Metallurgy at the Danish Engineering College indicated that the ball was made from 99% forged iron (Buchwald 1992, pers. comm.). Historical sources first mention firearms in Denmark in 1360 (Orloff, pers. comm.). The caliber, and the fact that the ball did not exit the skull, i.e. had a low muzzle velocity, indicates that the ball probably was fired from a small cannon (a so-called "skanseboesse"). This is the earliest firearms victim found in Denmark. Grave 202: The skeleton of an approx. 25 years old female yielded an unusal curved vertebral column (fig. 7) and bones with significantly thinner cortical bone mass. Our tentative conclusion is that the woman suffered from tuberculosis. Tuberculosis has been reported both in historical sources and from other Medieval excavations (Møller-Christensen 1958). Conclusion The preliminary results from the excavation of the St. Peder parish cemetery in Randers, show a Medieval (skeletal) population not differing much from other contemporary finds. The results have already formed part of other studies (Hjalgrim 1992). We expect to commence a research programme into Medieval paleothology in cooperation with the Smithsonian Institution of Washington, D.C., U.S.A., and the University of Bradford, England. The Randers skeletal material will then be a valuable contribution. B. Frøhlich, H. Hjalgrim,  J. Littleton, N. Lynnerup og B. Sejrsen.                      &nbsp