DNA repair (DNA Repair )

Publisher: Elsevier


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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively.
    DNA repair 08/2014;
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    ABSTRACT: Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. Mechanisms of TLS are lesion- and species-specific, with a prominent role of specialized DNA polymerases with relaxed active sites. After nucleotide(s) are incorporated across from the altered base(s), the aberrant primer termini are typically extended by DNA polymerase ζ (pol ζ). As a result, pol ζ is responsible for most DNA damage-induced mutations. The mechanisms of sequential DNA polymerase switches in vivo remain unclear. The major replicative DNA polymerase δ (pol δ) shares two accessory subunits, called Pol31/Pol32 in yeast, with pol ζ. Inclusion of Pol31/Pol32 in the pol δ/pol ζ holoenzymes requires a [4Fe-4S] cluster in C-termini of the catalytic subunits. Disruption of this cluster in Pol ζ or deletion of POL32 attenuates induced mutagenesis. Here we describe a novel mutation affecting the catalytic subunit of pol ζ, rev3ΔC, which provides insight into the regulation of pol switches. Strains with Rev3ΔC, lacking the entire C-terminal domain and therefore the platform for Pol31/Pol32 binding, are partially proficient in Pol32-dependent UV-induced mutagenesis. This suggests an additional role of Pol32 in TLS, beyond being a pol ζ subunit, related to pol δ. In search for members of this regulatory pathway, we examined the effects of Maintenance of Genome Stability 1 (Mgs1) protein on mutagenesis in the absence of Rev3-Pol31/Pol32 interaction. Mgs1 may compete with Pol32 for binding to PCNA. Mgs1 overproduction suppresses induced mutagenesis, but had no effect on UV-mutagenesis in the rev3ΔC strain, suggesting that Mgs1 exerts its inhibitory effect by acting specifically on Pol32 bound to pol ζ. The evidence for differential regulation of Pol32 in pol δ and pol ζ emphasizes the complexity of polymerase switches.
    DNA repair 05/2014;
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    ABSTRACT: A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit - searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair.
    DNA repair 05/2014;
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    ABSTRACT: Acetylation of α-tubulin on lysine 40 is one of the major posttranslational modifications of microtubules. The acetylation reaction is catalyzed by alpha-tubulin N-acetyltransferase and the modification can be reversed by either the NAD-independent class II histone deacetylase HDAC6 or the NAD-dependent deacetylase SIRT2. In this study, we assessed to what extent cellular NAD levels are involved in the regulation of the α-tubulin acetylation state. Cells were subjected to different treatments known to influence cellular NAD content. In response to NAD depletion caused by inhibition of NAD synthesis from nicotinamide, α-tubulin was hyperacetylated. Under these conditions, the normal tubulin acetylation state could be restored by providing the cells with alternative NAD precursors. Likewise, decreasing the rate of endogenous NAD consumption using an inhibitor of poly-ADP-ribosylation also stabilized the acetylation of α-tubulin. Conversely, the level of acetylated α-tubulin decreased when NAD synthesis was enhanced by overexpression of an NAD biosynthetic enzyme. Combined, these results show that the tubulin acetylation status is reciprocally regulated by cellular NAD levels. Furthermore, we provide evidence confirming that the NAD-dependent regulation of tubulin acetylation is mediated by SIRT2.
    DNA repair 05/2014;
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    ABSTRACT: The MutS2 homologues have been found widespread in most prokaryotes, which are involved in DNA repair and reactive oxygen species detoxification. The C-terminal small mutS-related (Smr) domain is critical for its endonucleolytic activity. However, the detailed catalytic mechanism is still unclear. In this study, we first investigated the in vivo role of drMutS2 in Deinococcus radiodurans, the most radiation-resistant organism exhibits the remarkable DNA repair capacity. mutS2 and recA mutS2 double knockout mutants were constructed because the phenotype was strongly masked by the predominant homologous recombination DNA repair pathway in this bacterium. Compared with the recA mutant, cells devoid of both genes showed increased sensitivity to ionizing radiation and oxidative agents, suggesting that drMutS2 is involved in RecA-independent mechanisms that enhance cellular resistance to oxidative stress-induced DNA damage. Moreover, the basal level of reductase activity and thiamine biosynthesis was induced in the absence of mutS2. To characterize its catalytic residues, the Smr domain was crystallized and soaked in buffer containing manganese ions. In contrast to native crystals, the space group of manganese-derivative crystals transformed from monoclinic to orthorhombic unexpectedly. This type of crystals showed improved diffraction resolution to 1.2Å, which has the highest resolution of currently known Smr structures. Structural comparison revealed that three acidic amino-acid residues, which are all located in the α1 helix, changed the rotamer states after metal soaking. Mutational analysis of conserved residue glutamic acid 710 to alanine yielded a drMutS2 variant with impaired nuclease activity, and could only partially rescue the radiosensitive phenotype of the mutS2 null strain, indicating that glutamic acid 710 is the catalytic residue.
    DNA repair 05/2014;
  • DNA repair 05/2014;
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    ABSTRACT: DNA non-homologous end-joining (NHEJ) is the major DNA double strand break (DSB) repair pathway in mammalian cells. Defects in NHEJ proteins confer marked radiosensitivity in cell lines and mice models, since radiation potently induces DSBs. The process of V(D)J recombination functions during the development of the immune response, and involves the introduction and rejoining of programmed DSBs to generate an array of diverse T and B cells. NHEJ rejoins these programmed DSBs. Consequently, NHEJ deficiency confers (severe) combined immunodeficiency – (S)CID – due to a failure to carry out V(D)J recombination efficiently. NHEJ also functions in class switch recombination, another step enhancing T and B cell diversity. Prompted by these findings, a search for radiosensitivity amongst (S)CID patients revealed a radiosensitive sub-class, defined as RS-SCID. Mutations in NHEJ genes, defining human syndromes deficient in DNA ligase IV (LIG4 Syndrome), XLF-Cernunnos, Artemis or DNA-PKcs, have been identified in such patients. Mutations in XRCC4 or Ku70,80 in patients have not been identified. RS-SCID patients frequently display additional characteristics including microcephaly, dysmorphic facial features and growth delay. Here, we overview the clinical spectrum of RS-SCID patients and discuss our current understanding of the underlying biology.
    DNA repair 05/2014;
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    ABSTRACT: DNA double strand breaks (DSBs) are a particularly cytotoxic variety of DNA lesion that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). HR utilises sequences homologous to the damage DNA template to facilitate repair. In contrast, NHEJ does not require homologous sequences for repair but instead functions by directly re-joining DNA ends. These pathways are critical to resolve DSBs generated intentionally during processes such as meiotic and site-specific recombination. However, they are also utilised to resolve potentially pathological DSBs generated by mutagens and errors during DNA replication. The importance of DSB repair is underscored by the findings that defects in these pathways results in chromosome instability that contributes to a variety of disease states including malignancy. The general principles of NHEJ are conserved in eukaryotes. As such, relatively simple model organisms have been instrumental in identifying components of these pathways and providing a mechanistic understanding of repair that has subsequently been applied to vertebrates. However, certain components of the NHEJ pathway are absent or show limited conservation in the most commonly used invertebrate models exploited to study DNA repair. Recently, however, it has become apparent that vertebrate DNA repair pathway components, including those involved in NHEJ, are unusually conserved in the amoeba Dictyostelium discoideum. Traditionally, this genetically tractable organism has been exploited to study the molecular basis of cell type specification, cell motility and chemotaxis. Here we discuss the use of this organism as an additional model to study DNA repair, with specific reference to NHEJ.
    DNA repair 05/2014;
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    ABSTRACT: Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1-/-) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1-/- cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1-/- cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.
    DNA repair 04/2014;
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    ABSTRACT: The emergence of high density technologies monitoring the genome, transcriptome and proteome in relation to genotoxic stress have tremendously enhanced our knowledge on global responses and dynamics in the DNA damage response, including its relation with cancer and aging. Moreover, '-omics' technologies identified many novel factors, their post-translational modifications, pathways and global responses in the cellular response to DNA damage. Based on omics, it is currently estimated that thousands of gene(product)s participate in the DNA damage response, recognizing complex networks that determine cell fate after damage to the most precious cellular molecule, DNA. The development of next generation sequencing technology and associated specialized protocols can quantitatively monitor RNA and DNA at unprecedented single nucleotide resolution. In this review we will discuss the contribution of omics technologies and in particular next generation sequencing to our understanding of the DNA damage response and the future prospective of next generation sequencing, its single cell application and omics dataset integration in unraveling intricate DNA damage signaling networks.
    DNA repair 04/2014;
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    ABSTRACT: While primordial life is thought to have been RNA-based (Cech, Cold Spring Harbor Perspect. Biol. 4 (2012) a006742), all living organisms store genetic information in DNA, which is chemically more stable. Distinctions between the RNA and DNA worlds and our views of "DNA" synthesis continue to evolve as new details emerge on the incorporation, repair and biological effects of ribonucleotides in DNA genomes of organisms from bacteria through humans.
    DNA repair 04/2014;