It is widely accepted that unrepaired or misrepaired DNA double strand breaks (DSBs) lead to the formation of chromosome aberrations. DSBs induced in the DNA of higher eukaryotes by endogenous processes or exogenous agents can in principle be repaired either by non-homologous endjoining (NHEJ), or homology directed repair (HDR). The basis on which the selection of the DSB repair pathway is made remains unknown but may depend on the inducing agent, or process. Evaluation of the relative contribution of NHEJ and HDR specifically to the repair of ionizing radiation (IR) induced DSBs is important for our understanding of the mechanisms leading to chromosome aberration formation. Here, we review recent work from our laboratories contributing to this line of inquiry. Analysis of DSB rejoining in irradiated cells using pulsed-field gel electrophoresis reveals a fast component operating with half times of 10–30 min. This component of DSB rejoining is severely compromised in cells with mutations in DNA-PKcs, Ku, DNA ligase IV, or XRCC4, as well as after chemical inhibition of DNA-PK, indicating that it reflects classical NHEJ; we termed this form of DSB rejoining D-NHEJ to signify its dependence on DNA-PK. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DSBs using an alternative pathway operating with slower kinetics (half time 2–10 h). This alternative, slow pathway of DSB rejoining remains unaffected in mutants deficient in several genes of the RAD52 epistasis group, suggesting that it may not reflect HDR. We proposed that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway. Biochemical studies confirm the presence in cell extracts of DNA end joining activities operating in the absence of DNA-PK and indicate the dominant role for D-NHEJ, when active. These observations in aggregate suggest that NHEJ, operating via two complementary pathways, B-NHEJ and D-NHEJ, is the main mechanism through which IR-induced DSBs are removed from the DNA of higher eukaryotes. HDR is considered to either act on a small fraction of IR induced DSBs, or to engage in the repair process at a step after the initial end joining. We propose that high speed D-NHEJ is an evolutionary development in higher eukaryotes orchestrated around the newly evolved DNA-PKcs and pre-existing factors. It achieves within a few minutes restoration of chromosome integrity through an optimized synapsis mechanism operating by a sequence of protein-protein interactions in the context of chromatin and the nuclear matrix. As a consequence D-NHEJ mostly joins the correct DNA ends and suppresses the formation of chromosome aberrations, albeit, without ensuring restoration of DNA sequence around the break. B-NHEJ is likely to be an evolutionarily older pathway with less optimized synapsis mechanisms that rejoins DNA ends with kinetics of several hours. The slow kinetics and suboptimal synapsis mechanisms of B-NHEJ allow more time for ...
We recently reported that two Chinese hamster mutants deficient in the RAD51 paralogs XRCC2 and XRCC3 show reduced radiosensitization after treatment with caffeine, thus implicating homology-directed repair (HDR) of DNA double-strand breaks (DSBs) in the mechanism of caffeine radiosensitization. Here, we investigate directly the effect of caffeine on HDR initiated by DSBs induced by a rare cutting endonuclease (I-SceI) into one of two direct DNA repeats. The results demonstrate a strong inhibition by caffeine of HDR in wild-type cells, and a substantial reduction of this effect in HDR-deficient XRCC3 mutant cells. Inhibition of HDR and cell radiosensitization to killing shows similar dependence on caffeine concentration suggesting a cause-effect relationship between these effects. UCN-01, a kinase inhibitor that effectively abrogates checkpoint activation in irradiated cells, has only a small effect on HDR, indicating that similar to radiosensitization, inhibition of checkpoint signaling is not sufficient for HDR inhibition. Recombination events occurring during treatment with caffeine are characterized by rearrangements reminiscent to those previously reported for the XRCC3 mutant, and immunofluorescence microscopy demonstrates significantly reduced formation of IR-specific RAD51 foci after caffeine treatment. In summary, our results identify inhibition of HDR as a significant contributor to caffeine radiosensitization.
A reliable autofocus is necessary for any combined image processing and automated microscope system analyse scan areas larger than a single field. Autofocus functions for brightfield microscopy have been reported in the literature. Autofocus implementations for fluorescence microscopy have to deal with some technical difficulties mainly due to the incoherence and faintness ofthe fluorescenct light. In this presentation autofocus procedures are introduced for fluorescence microscopy based on image content information. A Leitz MPV II fluorescence microscope with x,y,z stepping motors was used as basic equipment. The microscope images were captured with an intensified target camera, digitized with a frame grabber and analysed with a PC. We have constructed a digital autofocus system as well as an analogue autofocus detector. In the case of digital autofocus, the frame grabber images were analysed while changing the z-position ofthe microscope slide. Three different focus functions were investigated: 1) Edge-fmding algorithms such as LoG and Canny operators. 2) Different autocorrelation algorithms.In contrast to the digital focus criteria evaluation we constructed an electronic board with several differentiators and integrators. This module is designed for high speed autofocusing and directly coupled to the output ofthe camera. It makes it possible to obtain a focus value for the current image within one video cycle. Furthermore, it is possible to process three different focus functions (weighted intensity, 1 derivative and 2' derivative) simultaneously. Measurements showed that the most suitable focus function was dependent on the kind of fluorescent objects analysed. The flexibility to select a certain focus function or a combination ofthese functions allows us to use the analogue detector for various cell types and fluorescent dyes.All methods and functions were tested for different situations (fluorescence beads as well as different kinds of cells) and the results compared.
Image segmentation is almost always a necessary step in image processing. The employed threshold algorithms are based on the detection of local minima in the gray level histograms ofthe entire image. In automatic cell recognition equipment, like chromosome analysis or micronuclei counting systems' , flexible and adaptive thresholds are required to consider variation in gray level intensities ofthe background and ofthe specimen. We have studied three different methods ofthreshold determination:1 . a statistical procedure, which uses the interclass entropy maximisation of the gray level histogram. The iterative algorithm can be used for multi-threshold segmentation. The contribution of iteration step i is 2'' number of thresholds; 2. a numerical approach, which detects local minima in the gray level histogram. The algorithm must be tailored and optimized for specific applications like cell recognition with two different thresholds for cell nuclei and cell cytoplasm segmentation;3 . an artificial neural network, which is trained with learning sets of image histograms and the corresponding interactively determined thresholds. We have investigated feed forward networks with one and two layers, respectively. The gray level frequencies are used as inputs for the net. The number of different thresholds per image determines the output channels;We have tested and compared these different threshold algorithms for practical use in fluorescence microscopy as well as in bright field microscopy. The implementation and the results are presented and discussed.
During the past decade 3-dimensional image processing has become an important key component in biological research mainly due to two different developments. The first is based on an optical instrument, the so-called confocal laser scanning microscope, allowing optical sectioning of the biological specimen. The second is a biological preparatory method, the so-called FISH-technique (Fluorescence-In-Situ-Hybridization), allowing labeling of certain cellular and sub-cellular compartments with highly specific fluorescent dyes. Both methods make it possible to investigate the 3-dimensional biological framework within cells and nuclei.Image acquisition with confocal laser scanning microscopy must deal with different limits of resolution along and across the optical axis. Although lateral resolution is about 0.7 times better than in non-confocal arrangements, axial resolution is more than 3-4 times poorer than that of the lateral (depending on the pinhole size). For 3D reconstruction it is desirable to improve axial resolution in order to provide nearly identical image information across the 3 dimensional specimen space. This presentation will give an overview of some of the most popular restoration and deblurring algorithms used in 3D image microscopy.After 3D image restoration, segmentation of certain details of the cell structure is usually the next step in image processing. We compared two different kinds of algorithms for segmentation of chromosome territories in interphase cell nuclei. One is based on Mathematical Morphology, the other on Split & Merge methods.The segmented image regions provided the basis for chromosome domain reconstruction as well as for regional localization for subsequent quantitative measurements. As a result the chromatin density within certain chromosome domains as well as some terminal DNA sequences (telomere signals) could be measured.
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