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Available from: Martijn S Luijsterburg,
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    • "DNA photosensitizers induce numerous lesions within the DNA including base lesions, SSBs and DSBs (Table 1) [2,3,11,36–39] at the same time minimizing the contribution of UV-type damage. Not only the presence of photosensitisers but also the laser power may greatly influence the findings [40] as discussed below. The caveat of using photosensitizers is that the repair of DNA lesions may be hindered by the presence of the DNA interchelator and the exact mode of action of the photosensitizers, following excitation, electron transfer etc., has yet to be fully characterized. "
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    ABSTRACT: The formation of DNA lesions poses a constant threat to cellular stability. Repair of endogenously and exogenously produced lesions has therefore been extensively studied, although the spatiotemporal dynamics of the repair processes has yet to be fully understood. One of the most recent advances to study the kinetics of DNA repair has been the development of laser microbeams to induce and visualize recruitment and loss of repair proteins to base damage in live mammalian cells. However, a number of studies have produced contradictory results that are likely caused by the different laser systems used reflecting in part the wavelength dependence of the damage induced. Additionally, the repair kinetics of laser microbeam induced DNA lesions have generally lacked consideration of the structural and chemical complexity of the DNA damage sites, which are known to greatly influence their reparability. In this review, we highlight the key considerations when embarking on laser microbeam experiments and interpreting the real time data from laser microbeam irradiations. We compare the repair kinetics from live cell imaging with biochemical and direct quantitative cellular measurements for DNA repair.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 05/2013; 756(1-2). DOI:10.1016/j.mrgentox.2013.05.006 · 3.68 Impact Factor
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    • "– Recognizes ds/ss DNA junctions [203], Holliday junctions [83] [149], G4 structures [53], high energy laser DNA damages [204] [205] and positively supercoiled DNA [159] – Forms t-loops [7,78] – Binds TERRA RNA [72] – Protects Holliday junctions from resolvases cleavage and from Werner-mediated resolution [83] [150] – Condenses DNA [63] – Introduction of positive supercoils around itself [63] – Binds better positively supercoiled than relaxed or negatively supercoiled DNA [159] – Stimulates ssDNA invasion into duplex DNA [63] [80] – Regulates the enzymatic activities of Werner [192] [206], MUS81 [207], DNA polymerase beta [208], and Apollo [159] TIN2, RAP1, POT1, Apollo, ATM, MRN complex, WRN, BLM, Ku70, ORC1 and PARP1, 2 [195], ERCC1/XPF [209], Topoisomerase III [210], FEN1 and DNA polymerase beta [208], SLX4 [211] [212], REST [213], PNUTS and MCPH1 [214], MUS81/EME1 [207] – Telomere protection [77] – Replication of the EB virus episome [215] [216] and of telomeres [159] – Neuronal gene silencing [213] – Global repair? [204] Vertebrate RAP1 Repressor activator protein 1 – ScRap1 homolog contains a Myb-like (pdb 1FEX), a BRCT and a RCT domain – Does not bind DNA – Inhibits NHEJ-mediated ligation in vitro [217– 219] TRF2 [141], RAD50/MRE11 and KU86 [220], IKKs [221] – Telomere length regulation [220] [222] – Inhibition of NHEJ in human cells [219] – Inhibition of T-SCE in mice [223] – Participates in subtelomeric silencing and transcriptional regulation in mice [224] – Regulates NFjB-dependent gene expression [221] "
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    ABSTRACT: A major issue in telomere research is to understand how the integrity of chromosome ends is controlled. Although several nucleoprotein complexes have been described at the telomeres of different organisms, it is still unclear how they confer a structural identity to chromosome ends in order to mask them from DNA repair and to ensure their proper replication. In this review, we describe how telomeric nucleoprotein complexes are structured, comparing different organisms and trying to link these structures to telomere biology. It emerges that telomeres are formed by a complex and specific network of interactions between DNA, RNA and proteins. The fact that these interactions and associated activities are reinforcing each other might help to guaranty the robustness of telomeric functions across the cell cycle and in the event of cellular perturbations. We propose that telomeric nucleoprotein complexes orient cell fate through dynamic transitions in their structures and their organization.
    FEBS letters 09/2010; 584(17):3785-99. DOI:10.1016/j.febslet.2010.08.004 · 3.17 Impact Factor
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    • "Koenig et al. observed a reduction of the ablation threshold by a factor of 2.7 when single chromosomes were labeled with Giemsa [2]. In addition, four [33] to ten times [34] more pulse energy was required to induce DSBs in cells without Hoechst staining. Consequently, dye molecules significantly contribute to the production of free electrons and hence the formation of low-density plasmas. "
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    ABSTRACT: Femtosecond (fs) laser-based intracellular nanosurgery has become an important tool in cell biology, albeit the mechanisms in the so-called low-density plasma regime are largely unknown. Previous calculations of free-electron densities for intracellular surgery used water as a model substance for biological media and neglected the presence of dye and biomolecules. In addition, it is still unclear on which time scales free-electron and free-radical induced chemical effects take place in a cellular environment. Here, we present our experimental study on the influence of laser parameters and staining on the intracellular ablation threshold in the low-density plasma regime. We found that the ablation effect of fs laser pulse trains resulted from the accumulation of single-shot multiphoton-induced photochemical effects finished within a few nanoseconds. At the threshold, the number of applied pulses was inversely proportional to a higher order of the irradiance, depending on the laser repetition rate and wavelength. Furthermore, fluorescence staining of subcellular structures before surgery significantly decreased the ablation threshold. Based on our findings, we propose that dye molecules are the major source for providing seed electrons for the ionization cascade. Consequently, future calculations of free-electron densities for intracellular nanosurgery have to take them into account, especially in the calculations of multiphoton ionization rates.
    Biomedical Optics Express 09/2010; 1(2):587-597. DOI:10.1364/BOE.1.000587 · 3.65 Impact Factor
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