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[en] We report on a novel resonant THz sensor for the label-free analysis of DNA molecules. The sensor allows the direct detection of DNA-probe molecules at functionalized electrodes via hybridization. Subsequent time resolved photoconductive sampling of the THz transmission identifies the binding state between probe and target DNA. Integrating neighbouring sensors on a chip, this technique can be extended to a parallel analysis of multiple DNA sequences. A clearly readable sensor response is obtained with less then 40 fmol of 20-mer single-stranded DNA molecules, indicating at least a sevenfold sensitivity increase compared to previous approaches
[en] Highlights: • Mitochondria and mtDNA are heterogeneous under many aspects. • MtDNA replication requires mitochondrial and nuclear factors. • Threats to mtDNA replication arise from the structure/organization of this genome. • Sub-optimal mtDNA replication is associated with disease. • The mtDNA content is not necessarily associated to the mitochondrial mass. - Abstract: Mitochondrial DNA (mtDNA), which is essential for mitochondrial and cell function, is replicated and transcribed in the organelle by proteins that are entirely coded in the nucleus. Replication of mtDNA is challenged not only by threats related to the replication machinery and orchestration of DNA synthesis, but also by factors linked to the peculiarity of this genome. Indeed the architecture, organization, copy number, and location of mtDNA, which are markedly distinct from the nuclear genome, require ad hoc and complex regulation to ensure coordinated replication. As a consequence sub-optimal mtDNA replication, which results from compromised regulation of these factors, is generally associated with mitochondrial dysfunction and disease. Mitochondrial DNA replication should be considered in the context of the organelle and the whole cell, and not just a single genome or a single replication event. Major threats to mtDNA replication are linked to its dependence on both mitochondrial and nuclear factors, which require exquisite coordination of these crucial subcellular compartments. Moreover, regulation of replication events deals with a dynamic population of multiple mtDNA molecules rather than with a fixed number of genome copies, as it is the case for nuclear DNA. Importantly, the mechanistic aspects of mtDNA replication are still debated. We describe here major challenges for human mtDNA replication, the mechanistic aspects of the process that are to a large extent original, and their consequences on disease.
[en] BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair
[en] The examination and profiling of human DNA recovered from a scene of crime is an essential aspect of criminal investigations. However, it is currently not known whether DNA recovered from a scene where an ionising radiation source or radioactive contamination is present can be successfully profiled. The direct examination and analysis of radioactively contaminated DNA has not been widely explored using the current procedures employed by forensic service providers. As a result, AWE is putting in place an extensive research and development programme to better understand the effects that radiation has on the ability to profile human DNA, and assess the associated retention of different radioactive contaminants within each step of the profiling procedure. A summary will be provided on the aims of this project and progress that has been made to date; together with a discussion of the lessons that have been learnt during the course of the programme's development. (author)
[en] Nick translation, or more precisely nick translocation, is a specific procedure for incorporating radioactive nucleotides into double-stranded DNA. The method takes advantage of the ability of Escherichia coli DNA polymerase I to combine the sequential addition of nucleotide residues to the 3'-hydroxyl terminus of a nick with the elimination of nucleotides from the adjacent 5'-phosphoryl terminus. Linear, supercoiled, nicked, or gapped circular double-stranded molecules can be labeled to specific activities > 108 cpm/μg with deoxynucleotide 5'-[α-32P]triphosphates by this technique. Since the nicks are introduced at random sites in the duplex, the method generates a population of radioactive fragments which partially overlap each other. At saturating levels of nucleotide triphosphates the size of the fragments is determined by the DNase concentration. While experiments consistent with hyperpolymer formation of nick-translated probes have been reported, the reproducibility and extent of hyperpolymer formation seem to be difficult to obtain, probably because of the critical dependence on probe size
[en] Replication stress is a strong and early driving force for genomic instability and tumor development. Beside replicative DNA polymerases, an emerging group of specialized DNA polymerases is involved in the technical assistance of the replication machinery in order to prevent replicative stress and its deleterious consequences. During S-phase, altered progression of the replication fork by endogenous or exogenous impediments induces replicative stress, causing cells to reach mitosis with genomic regions not fully duplicated. Recently, specific mechanisms to resolve replication intermediates during mitosis with the aim of limiting DNA damage transmission to daughter cells have been identified. In this review, we detail the two major actions of specialized DNA polymerases that limit DNA damage transmission: the prevention of replicative stress by non-B DNA replication and the recovery of stalled replication forks.
[en] DNA mismatch repair (MMR) is critical for the maintenance of genomic stability. MMR is initiated by recognition of DNA mismatches by the protein, MutS, which subsequently recruits downstream repair factors. To better understand the mechanism by which MutS identifies and specifically binds mismatched basepairs embedded in random DNA sequences, we monitored the interaction between MutS and DNA substrates using atomic force microscopy (AFM). An α-shaped DNA loop formed by the interaction between MutS and DNA, which was independent of whether or not a mismatch was present in the DNA substrate. These data indicate that MutS associates with DNA non-specifically and forms an α-loop interaction with the DNA substrate. In this conformation, MutS is able to scan two arms of DNA simultaneously for each MutS dimer formed
[en] DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Eschrichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the [3H]-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of th segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. To determine the number of replications of pSW14, a novel method was employed. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated [3H]thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude the pSW14 has a functional origin in the Xenopus DNA segment
[en] Interlinear dimers are complex-type lesions, and both DNA strands are covalently bridged. Therefore, this type of the lesion is very toxic to the cell because it interferes with the separation of strands of DNA required for crucial processes for the cell, such as the replication and transcription. In addition, recent experiences show that the repair of inter-flaming dimeras involves training a double-strand break, another lesion with a high toxic potential. It is only recently that we have shown that ionizing radiation led to the formation of interlinear dimeras in cellular DNA. We still knows very little about the conditions in which the dimers occur and how they are repaired. .Recently, following a exposure to ionizing radiation, our group highlighted the formation of interlinear dimer in a DNA or thymidine had been replaced by 5-bromo-2'-desoxyuridine (BrdU). These dimeres were not only when the BrdU was in the center of a mapped area. Since this was the first time this type of damage was observed during exposure of brominated DNA to ionizing radiation, my thesis deals with the exploration of the formation of the interbriniferous dimeras particularly on the conditions which favored its formation. The three papers presented in this thesis show that the form of DNA (form A vs form B), the sequence, as well as the type of radiation employed have a significant influence on the type and frequency of product damage. These results show that we still know very little about the actual mechanism for radiosensitizing of bromine DNA in cells. However, they also highlight the distinct reactivity regions of their DNA, as well as their high potential for formation of dimeres. However, these mapped regions represent only one fraction of the secondary and tertiary structures of DNA present in the cell.
[en] Radiation can damage cellular components, including DNA. Organisms have developed a panoply of means of dealing with DNA damage. Some repair paths have rather narrow substrate specificity (e.g. photolyases), which act on specific pyrimidine photoproducts in a specific type (e.g., DNA) and conformation (double-stranded B conformation) of nucleic acid. Others, for example, nucleotide excision repair, deal with larger classes of damages, in this case bulky adducts in DNA. A detailed discussion of DNA repair mechanisms is beyond the scope of this article, but one can be found in the excellent book of Friedberg et al. for further detail. However, some DNA damages and paths for repair of those damages important for photobiology will be outlined below as a basis for the specific examples of genetic and molecular analysis that will be presented below