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  • CRISPR/CAS9-based DNA damage response screens reveal gene-drug interactions
    DNA Repair (IF 3.711) Pub Date : 2020-01-16
    Dan Su; Xu Feng; Medina Colic; Yunfei Wang; Chunchao Zhang; Chao Wang; Mengfan Tang; Traver Hart; Junjie Chen

    DNA damage response (DDR) is critically important for cell survival, genome maintenance, and its defect has been exploited therapeutically in cancer treatment. Many DDR-targeting agents have been generated and have entered the clinic and/or clinical trials. In order to provide a global and unbiased view of DDR network, we designed a focused CRISPR library targeting 365 DDR genes and performed CRISPR screens on the responses to several DDR inhibitors and DNA-damaging agents in 293A cells. With these screens, we determined responsive pathways enriched under treatment with different types of small-molecule agents. Additionally, we showed that POLE3/4-deficient cells displayed enhanced sensitivity to an ATR inhibitor, a PARP inhibitor, and camptothecin. Moreover, by performing DDR screens in isogenic TP53 wild-type and TP53 knock-out cell lines, our results suggest that the performance of our CRISPR DDR dropout screens is independent of TP53 status. Collectively, our findings indicate that CRISPR DDR screens can be used to identify potential targets of small-molecule drugs and reveal that TP53 status does not affect the outcome of these screens.

    更新日期:2020-01-17
  • HPV induction of APOBEC3 enzymes mediate overall survival and response to cisplatin in head and neck cancer
    DNA Repair (IF 3.711) Pub Date : 2020-01-16
    Kayla L. Conner; Asra N. Shaik; Elmira Ekinci; Seongho Kim; Julie J. Ruterbusch; Michele L. Cote; Steve M. Patrick

    Human papillomavirus (HPV) is associated with the development of head and neck squamous cell carcinomas (HNSC). Cisplatin is used to treat HNSC and induces DNA adducts including interstrand crosslinks (ICLs). Previous reports have shown that HPV positive HNSC patients respond better to cisplatin therapy. Our previous reports highlight that loss of base excision repair (BER) and mismatch repair (MMR) results in cisplatin resistance. Of importance, uracil DNA glycosylase (UNG) is required to initiate the BER response to cisplatin treatment and maintain drug sensitivity. These previous results highlight that specific cytidine deaminases could play an important role in the cisplatin response by activating the BER pathway to mediate drug sensitivity. The APOBEC3 (A3) family of cytidine deaminases are enzymes that restrict HPV as part of the immune defense to viral infection. In this study, the Cancer Genome Atlas (TCGA) HNSC data were used to assess the association between the expression of the seven proteins in the A3 cytidine deaminase family, HPV-status and survival outcomes. Higher A3 G expression in HPV-positive tumors corresponds with better overall survival (OS) (HR 0.33, 95% CI 0.11-0.93, p = 0.04). FaDu and Scc-25 HNSC cell lines were used to assess alterations in A3, BER and MMR expression in response to cisplatin. We demonstrate that A3, Polβ, and MSH6 knockdown in HNSC cells results in resistance to cisplatin and carboplatin as well as an increase in the rate of ICL removal in FaDu and Scc-25 HNSC cells. Our results suggest that A3s activate BER in HNSC, mediate repair of cisplatin ICLs and thereby, sensitize cells to cisplatin which likely contributes to the improved patient responses observed in HPV infected patients.

    更新日期:2020-01-16
  • Oxidative DNA-protein Crosslinks Formed in Mammalian Cells by Abasic Site Lyases Involved in DNA Repair
    DNA Repair (IF 3.711) Pub Date : 2020-01-09
    Jason L. Quiñones; Upasna Thapar; Samuel H. Wilson; Dale A. Ramsden; Bruce Demple

    Free radical attack on C1′ of deoxyribose forms the oxidized abasic (AP) site 2-deoxyribonolactone (dL). In vitro, dL traps the major base excision DNA repair enzyme DNA polymerase beta (Polβ) in covalent DNA-protein crosslinks (DPC) via the enzyme’s N-terminal lyase activity acting on 5′-deoxyribose-5-phosphate residues. We previously demonstrated formation of Polβ-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Polβ-DPC accumulation and trapped ubiquitin in the DPC, with Polβ accounting for 60-70% of the total ubiquitin signal. However, the identity of the remaining oxidative ubiquityl-DPC remained unknown. In this report, we surveyed whether additional AP lyases are trapped in oxidative DPC in mammalian cells in culture. Poly(ADP-ribose) polymerase 1 (PARP1), Ku proteins, DNA polymerase λ (Polλ), and the bifunctional 8-oxoguanine DNA glycosylase 1 (OGG1), were all trapped in oxidative DPC in mammalian cells. We also observed significant trapping of Polλ, PARP1, and OGG1 in cells treated with the alkylating agent methylmethane sulfonate (MMS), in addition to dL-inducing agents. Ku proteins, in contrast, followed a pattern of trapping similar to that for Polβ: MMS failed to produce Ku-DPC, while treatment with CuOP or (less effectively) H2O2 gave rise to significant Ku-DPC. Unexpectedly, NEIL1 and NEIL3 were trapped following H2O2 treatment, but not detectably in cells exposed to CuOP. The half-life of all the AP lyase-DPC ranged from 15-60 min, consistent with their active repair. Accordingly, CuOP treatment under proteasome inhibition significantly increased the observed levels of DPC in cultured mammalian cells containing PARP1, Ku protein, Polλ, and OGG1 proteins. As seen for Polβ, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Polβ-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells.

    更新日期:2020-01-09
  • The mismatch repair protein MSH6 regulates somatic recombination in Arabidopsis thaliana
    DNA Repair (IF 3.711) Pub Date : 2020-01-09
    Valentina Gonzalez; Claudia P. Spampinato

    The mismatch repair (MMR) pathway promotes genome stability by controlling the fidelity of replication and recombination. The first step of the pathway involves recognition of the mismatch by heterodimers composed of MutS homologs (MSH). Although MSH6 has been well characterized in yeasts and humans, the role of the plant protein has not been extensively studied. We first analyzed gene expression in Arabidopsis thaliana. The use of transgenic plants expressing the β-glucuronidase (GUS) reporter gene under the control of approximately 1-kb region upstream of the start codon of the AtMSH6 gene demonstrated that MSH6 is preferentially expressed in undifferentiated cells with an intense cell division rate. We then examined protein function in meiotic and somatic recombination. Suppression of AtMSH6 did not affect the rate of meiotic recombination, but increased the frequency of recombination between two homeologous repeats of a marker gene by 3-fold relative to wild-type plants. Expression of the AtMSH6 gene under the control of its own promoter in msh6 homozygous mutant plants rescued the altered somatic recombination phenotype. We conclude that MSH6 shows a functional conservation across different biological kingdoms and a functional specificity in plants.

    更新日期:2020-01-09
  • Repair of DNA-protein crosslinks in plants
    DNA Repair (IF 3.711) Pub Date : 2020-01-08
    Leonie Hacker; Annika Dorn; Holger Puchta

    DNA-protein crosslinks represent a severe kind of DNA damage as they disturb essential processes, such as transcription and DNA replication, due to their bulkiness. To ensure the maintenance of genome integrity, it is necessary for all living organisms to repair these lesions in a timely manner. Over recent years, much knowledge has been obtained regarding the repair of DNA-protein crosslinks (DPC), but it was only recently that the first insights into the mechanisms of DPC repair in plants were obtained. The plant DPC repair network consists of at least three parallel pathways that resolve DPC by distinct biochemical mechanisms. The endonuclease MUS81 resolves the DPC by cleaving the DNA part of the crosslink, the protease WSS1A is able to degrade the protein part and the tyrosyl-DNA-phosphodiesterase TDP1 can hydrolyse the crosslink between a protein and the DNA. However, due to the variety of different DPC types and the evolutionary conservation of pathways between eukaryotes, we expect that future research will reveal additional factors involved in DPC repair in plants.

    更新日期:2020-01-09
  • DNA-protein crosslink repair in plants
    DNA Repair (IF 3.711) Pub Date : 2020-01-07
    Leonie Hacker; Annika Dorn; Holger Puchta

    DNA-protein crosslinks represent a severe kind of DNA damage as they disturb essential processes like transcription and DNA replication, due to their bulkiness. To ensure the maintenance of genome integrity, it is necessary for all living organisms to repair these lesions in a timely manner. Over the last years, much knowledge has been obtained regarding the repair of DNA-protein crosslinks (DPCs), however it was only recently that the first insights into the mechanisms of DPC repair in plants were observed. The plant DPC network consists of at least three parallel pathways that resolve DPCs in distinct biochemical ways. The endonuclease MUS81 resolves the DPC by cleaving the DNA part of the crosslink, the protease WSS1A is able to degrade the protein part and the tyrosyl-DNA-phosphodiesterase TDP1 can hydrolyse the crosslink between a protein and the DNA. However, due to the variety of different DPC types and the evolutionary conservation of pathways between eukaryotes, we expect that future research will reveal additional factors involved in DPC repair in plants.

    更新日期:2020-01-07
  • The activity of the DNA repair enzyme hOGG1 can be directly modulated by ubiquinol
    DNA Repair (IF 3.711) Pub Date : 2020-01-03
    Daniel Schniertshauer; Daniel Gebhard; Heiko van Beek; Vivien Nöth; Julia Schon; Jörg Bergemann

    The DNA of human cells suffers about 1.000 to 100.000 oxidative lesions per day. One of the most common defects in this category is represented by 7,8-dihydro-8-oxoguanine. There are numerous exogenous effects on DNA that induce the intracellular generation of 7, 8-dihydro-8-oxoguanine. Therefore, a quantitatively sufficient repair of all occurring oxidative damaged guanine bases is often only partially feasible, especially in advanced age. Inadequate removal of these damages can subsequently lead to mutations and thus to serious diseases. All these aspects represent a dangerous situation for an organism. However, it is suspected that the amount of the 8-oxoguanine DNA glycosylase can be actively regulated on the level of gene expression by the redox-active properties of ubiquinol and thus its protein expression can be controlled. Using an real-time base excision repair assay including a melting curve analysis, the activity of the human 8-oxoguanine DNA glycosylase 1 was measured under the influence of ubiquinol. It was possible to observe a concentration-dependent increase in the activity of the 8-oxoguanine DNA glycosylase 1 under the influence of ubiquinol for the first time, both on purified and commercially acquired enzyme as well as on enzyme isolated from mitochondria of human fibroblasts. An increase in activity of this enzyme based on a change in cellular redox state caused by ubiquinol could not be confirmed. In addition, an increased gene expression of 8-oxoguanine-DNA glycosylase 1 under ubiquinol could not be observed. However, there was a change in bifunctionality in favor of an increased N-glycosylase activity and a direct interaction between ubiquinol and 8-oxoguanine DNA glycosylase 1. We suggest that ubiquinol contributes to the dissolution of a human 8-oxoguanine DNA glycosylase 1 end-product complex that forms after cutting into the sugar-phosphate backbone of the DNA with the resulting unsaturated 3'-phospho-α, β-aldehyde end and thereby inhibits further enzymatic steps.

    更新日期:2020-01-04
  • 更新日期:2019-12-29
  • Genotoxic Effects of Topoisomerase Poisoning and PARP inhibition on Zebrafish Embryos
    DNA Repair (IF 3.711) Pub Date : 2019-12-19
    Margarita Karapetian; Sophiko Tsikarishvili; Nina Kulikova; Anna Kurdadze; Giorgi Zaalishvili

    Topoisomerase poisons are known to stabilize covalent enzyme-DNA intermediates forming covalent cleavage complexes, which are highly cytotoxic especially for dividing cells and thus, make topoisomerases targets for cancer therapy. Topoisomerases have been extensively studied in mammalian model systems, whereas in other vertebrate models including zebrafish, they still remain less characterized. Here we show similarities in the genotoxic effects of zebrafish and mammalian systems towards topoisomerase I (Top1) poisons and PARP inhibitor - olaparib. On the other hand we observed that topoisomerase II (Top2) poisons (doxorubicin and etoposide) did not affect 1 day post fertilization embryo viability, however in cells isolated from Top2 drug treated embryos the formation of DNA cleavage complexes was observed by comet assay. We explain this by cellular drug uptake limitation in live zebrafish embryos versus unimpeded drug influx in cells isolated from Top2 poisons pre-treated embryos. We also demonstrate that EDTA facilitates the extraction of Top2 from zebrafish nuclei and recovers both, basal and Top2 poison induced DNA damage.

    更新日期:2019-12-19
  • Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts
    DNA Repair (IF 3.711) Pub Date : 2019-12-14
    Jun-ichi Akagi; Keiji Hashimoto; Kenji Suzuki; Masayuki Yokoi; Niels de Wind; Shigenori Iwai; Haruo Ohmori; Masaaki Moriya; Fumio Hanaoka
    更新日期:2019-12-17
  • The splicing component ISY1 regulates APE1 in base excision repair
    DNA Repair (IF 3.711) Pub Date : 2019-12-13
    Aruna S. Jaiswal; Elizabeth A. Williamson; Gayathri Srinivasan; Kimi Kong; Carrie L. Lomelino; Robert Mckenna; Christi Walter; Patrick Sung; Satya Narayan; Robert Hromas

    The integrity of cellular genome is continuously challenged by endogenous and exogenous DNA damaging agents. If DNA damage is not removed in a timely fashion the replisome may stall at DNA lesions, causing fork collapse and genetic instability. Base excision DNA repair (BER) is the most important pathway for the removal of oxidized or mono-alkylated DNA. While the main components of the BER pathway are well defined, its regulatory mechanism is not yet understood. We report here that the RNA splicing factor ISY1 enhances apurinic/apyrimidinic endonuclease 1 (APE1) activity, the multifunctional enzyme in BER, by promoting its 5’-3’ endonuclease activity. ISY1 expression is induced by oxidative damage, which would provide an immediate up-regulation of APE1 activity in vivo and enhance BER of oxidized bases. We further found that APE1 and ISY1 interact, and ISY1 enhances the ability of APE1 to recognize abasic sites in DNA. Using purified recombinant proteins, we reconstituted BER and demonstrated that ISY1 markedly promoted APE1 activity in both the short- and long-patch BER pathways. Our study identified ISY1 as an important regulator of the BER pathway, which would be of physiological relevance where suboptimal levels of APE1 are present. The interaction of ISY1 and APE1 also establishes a connection between DNA damage repair and pre-mRNA splicing.

    更新日期:2019-12-13
  • Genetic diversity and functional effect of common polymorphisms in genes involved in the first heterodimeric complex of the Nucleotide Excision Repair pathway
    DNA Repair (IF 3.711) Pub Date : 2019-12-12
    Yosr Hamdi; Manel Jerbi; Lilia Romdhane; Mariem Ben Rekaya; Houda El Benna; Lotfi Chouchane; Mohamed Samir Boubaker; Sonia Abdelhak; Houda Yacoub-Youssef

    Nucleotide excision repair is a multistep process that recognizes and eliminates a spectrum of DNA damages. Five proteins, namely XPC, RAD23, Centrin2, DDB1 and DDB2 act as a heterodimeric complex at the early steps of the NER pathway and play a crucial role in the removal of DNA lesions. Several exonic mutations on genes coding for these proteins have been identified as associated with Xeroderma-pigmentosum (XP), a rare monogenic disorder. However, the role of regulatory polymorphisms in disease development and inter-ethnic diversity is still not well documented. Due to the high incidence rate of XP in Tunisia, we performed a genotyping analysis of 140 SNPs found on these 5 genes in a set of 135-subjects representing the general Tunisian-population. An inter-ethnic comparison based on the genotype frequency of these SNPs have been also conducted. For the most relevant variants, we performed a comprehensive assessment of their functional effects. Linkage disequilibrium and principal component analysis showed that the Tunisian-population is an admixed and intermediate population between Sub-Saharan Africans and Europeans. Using variable factor maps, we identified a list of 20 polymorphisms that contribute considerably to the inter-ethnic diversity of the NER complex. In-silico functional analysis showed that SNPs on XPC, DDB1 and DDB2 are associated with eQTLs mainly DDB2-rs10838681 that seems to decrease significantly the expression level of ACP2 (p = 6.1 × 10-26). Statistical analysis showed that the allelic frequency of DDB2-rs10838681 in Tunisia is significantly different from all other populations. Using rVarBase, we identified 5 variants on XPC, DDB1 and DDB2 that seem to alter the binding sites of several transcription factors considered as key players in DNA-repair pathways. Results presented in this study provide the first report on regulatory polymorphisms of the NER-complex genes in Tunisia. These results may also help to establish a baseline database for future association and functional studies.

    更新日期:2019-12-13
  • MACROMOLECULAR CROWDING INDUCES COMPACTION AND DNA BINDING IN THE DISORDERED N-TERMINAL DOMAIN OF hUNG2
    DNA Repair (IF 3.711) Pub Date : 2019-12-10
    Gaddiel Rodriguez, Benjamin Orris, Ananya Majumdar, Shridhar Bhat, James T. Stivers
    更新日期:2019-12-11
  • Detection of the small oligonucleotide products of nucleotide excision repair in UVB-irradiated human skin
    DNA Repair (IF 3.711) Pub Date : 2019-12-05
    Jun-Hyuk Choi, Sueji Han, Michael G. Kemp
    更新日期:2019-12-05
  • Computational investigations and molecular dynamics simulations envisioned for potent antioxidant and anticancer drugs using indole-chalcone-triazole hybrids
    DNA Repair (IF 3.711) Pub Date : 2019-12-05
    Sanjai Kumar Yadav, Ramesh Kumar Yadav, Umesh Yadava

    Cancer, also called malignancy, is a disease which is closely related with the oxidative stress instigated by the overproduction of vulnerable oxygen and nitrogen species. Available drugs are relatively painful and toxic and so are trailing their captivation. Keeping this in mind, we have attempted to reach a novel anti-cancer drug by taking a set of nineteen ligands which are hybrids of Indole-chalcone and triazole. These ligands were allowed to interact with the DNA dodecamer 5ˊ(CGCGAATTCGCG)3ˊ one by one using various docking protocols of Glide. Better docked complexes screened through docking scores and reported activity data were selected and exposed to molecular dynamics run of 20 ns. The dynamical pathways were investigated for each complex comparing the pre- and post- dynamics run. The outcome of the work is discussed in this paper. Among the better hybrids of this series, one of the molecules has shown interesting features, confirming its non-toxic nature and working as intercalator as well minor groove binder, perhaps making it suitable as a potent drug for further pharmacological use.

    更新日期:2019-12-05
  • Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis
    DNA Repair (IF 3.711) Pub Date : 2019-12-05
    Alexandra M. Gehring, Kelly M. Zatopek, Brett W. Burkhart, Vladimir Potapov, Thomas J. Santangelo, Andrew F. Gardner

    Reactive oxygen species drive the oxidation of guanine to 8-oxoguanine (8oxoG), which threatens genome integrity. The repair of 8oxoG is carried out by base excision repair enzymes in Bacteria and Eukarya, however, little is known about archaeal 8oxoG repair. This study identifies a member of the Ogg-subfamily archaeal GO glycosylase (AGOG) in Thermococcus kodakarensis, an anaerobic, hyperthermophilic archaeon, and delineates its mechanism, kinetics, and substrate specificity. TkoAGOG is the major 8oxoG glycosylase in T. kodakarensis, but is non-essential. In addition to TkoAGOG, the major apurinic/apyrimidinic (AP) endonuclease (TkoEndoIV) required for archaeal base excision repair and cell viability was identified and characterized. Enzymes required for the archaeal oxidative damage base excision repair pathway were identified and the complete pathway was reconstituted. This study illustrates the conservation of oxidative damage repair across all Domains of life.

    更新日期:2019-12-05
  • Systematic Analysis of Linker Histone PTM Hotspots Reveals Phosphorylation Sites that Modulate Homologous Recombination and DSB Repair
    DNA Repair (IF 3.711) Pub Date : 2019-11-29
    Kuntal Mukherjee, Nolan English, Chance Meers, Hyojung Kim, Alex Jonke, Francesca Storici, Matthew Torres

    Double strand-breaks (DSBs) of genomic DNA caused by ionizing radiation or mutagenic chemicals are a common source of mutation, recombination, chromosomal aberration, and cell death. Linker histones are DNA packaging proteins with established roles in chromatin compaction, gene transcription, and in homologous recombination (HR)-mediated DNA repair. Using a machine-learning model for functional prioritization of eukaryotic post-translational modifications (PTMs) in combination with genetic and biochemical experiments with the yeast linker histone, Hho1, we discovered that site-specific phosphorylation sites regulate HR and HR-mediated DSB repair. Five total sites were investigated (T10, S65, S141, S173, and S174), ranging from high to low function potential as determined by the model. Of these, we confirmed S173/174 are phosphorylated in yeast by mass spectrometry and found no evidence of phosphorylation at the other sites. Phospho-nullifying mutations at these two sites results in a significant decrease in HR-mediated DSB repair templated either with oligonucleotides or a homologous chromosome, while phospho-mimicing mutations have no effect. S65, corresponding to a mammalian phosphosite that is conserved in yeast, exhibited similar effects. None of the mutations affected base- or nucleotide-excision repair, nor did they disrupt non-homologous end joining or RNA-mediated repair of DSBs when sequence heterology between the break and repair template strands was low. More extensive analysis of the S174 phospho-null mutant revealed that its repression of HR and DSB repair is proportional to the degree of sequence heterology between DSB ends and the HR repair template. Taken together, these data demonstrate the utility of machine learning for the discovery of functional PTM hotspots, reveal linker histone phosphorylation sites necessary for HR and HR-mediated DSB repair, and provide insight into the context-dependent control of DNA integrity by the yeast linker histone Hho1.

    更新日期:2019-11-30
  • A spontaneous mutation in DNA polymerase POL3 during in vitro passaging causes a hypermutator phenotype in Cryptococcus species
    DNA Repair (IF 3.711) Pub Date : 2019-11-29
    Kylie J. Boyce, Chengjun Cao, Chaoyang Xue, Alexander Idnurm

    Passaging of microbes in vitro can lead to the selection of microevolved derivatives with differing properties to their original parent strains. One well characterised instance is the phenotypic differences observed between the series of strains derived from the type strain of the human pathogenic fungus Cryptococcus neoformans. A second case was reported in the close relative Cryptococcus deneoformans, in which a well-studied isolate ATCC 24067 (52D) altered its phenotypic characteristics after in vitro passaging in different laboratories. One of these derivatives, ATCC 24067A, has decreased virulence and also exhibits a hypermutator phenotype, in which the mutation rate is increased compared to wild type. In this study, the molecular basis behind the changes in the lineage of ATCC 24067 was determined by next-generation sequencing of the parent and passaged strain genomes. This analysis resulted in the identification of a point mutation that causes a D270 G amino acid substitution within the exonuclease proofreading domain of the DNA polymerase delta subunit encoded by POL3. Complementation with POL3 confirmed that this mutation is responsible for the hypermutator phenotype of this strain. Regeneration of the mutation in C. neoformans to eliminate the additional mutations present in the genetic background, demonstrated that the hypermutator phenotype of the pol3D270G mutant causes rapid microevolution in vitro but does not result in decreased virulence. These findings indicate that mutator strains can emerge in these pathogenic fungi without conferring a fitness cost, but the subsequent rapid accumulation of mutations can be deleterious.

    更新日期:2019-11-30
  • Enhancement of DNA Damage Repair Potential in Germ Cells of Caenorhabditis elegans by a Volatile Signal from Their Irradiated Partners
    DNA Repair (IF 3.711) Pub Date : 2019-11-26
    Huangqi Tang, Liangwen Chen, Zhangyu Dai, Wenjing Zhang, Ting Wang, Lijun Wu, Gaohong Wang, Po Bian

    Radiation-induced bystander effects have been demonstrated within organisms. Recently, it is found that the organisms can also signal irradiation cues to their co-cultured partners in a waterborne manner. In contrast, there is a limited understanding of radiation-induced airborne signaling between individuals, especially on the aspect of DNA damage responses (DDR). Here, we establish a co-culture experimental system using Caenorhabdis elegans in a top-bottom layout, where communication between “top” and “bottom” worms is airborne. The radiation response of top worms is evaluated using radio-adaptive response (RAR) of embryonic lethality (F1), which reflects an enhancement in repair potential of germ cells to subsequent DNA damage. It is shown that gamma-irradiation of bottom worms alleviates the embryonic lethality of top worms caused by 25 Gy of subsequent gamma-irradiation, i.e. RAR, indicating that a volatile signal might play an essential role in radiation-induced inter-worm communication. The RAR is absent in the top worms impaired in DNA damage checkpoint, nucleotide excision repair, and olfactory sensory neurons, respectively. The induction of RAR is restricted to the mitotic zone of the female germline of hermaphrodites. These results indicate that the top worms sense the volatile signal through cephalic sensory neurons, and the neural stimulation distantly modulates the DDR in germ mitotic cells, leading to the enhancement of DNA damage repair potential. The volatile signal is produced specifically by the L3-stage bottom worms and functionally distinct from the known sex pheromone. Its production and/or release are regulated by water-soluble ascaroside pheromones in a population-dependent manner.

    更新日期:2019-11-27
  • Loss of Setd4 delays radiation-induced thymic lymphoma in mice
    DNA Repair (IF 3.711) Pub Date : 2019-11-25
    Xing Feng, Huimei Lu, Jingyin Yue, Neta Schneider, Jingmei Liu, Lisa K Denzin, Chang S. Chan, Subhajyoti De, Zhiyuan Shen

    Radiation-induced lymphomagenesis results from a clonogenic lymphoid cell proliferation due to genetic alterations and immunological dysregulation. Mouse models had been successfully used to identify risk and protective factors for radiation-induced DNA damage and carcinogenesis. The mammalian SETD4 is a poorly understood putative methyl-transferase. Here, we report that conditional Setd4 deletion in adult mice significantly extended the survival of radiation-induced T-lymphoma. However, in Tp53 deficient mice, Setd4 deletion did not delay the radiation-induced lymphomagenesis although it accelerated the spontaneous T-lymphomagenesis in non-irradiated mice. The T-lymphomas were largely clonogenic in both Setd4flox/flox and Setd4Δ/Δ mice based on sequencing analysis of the T-cell antigen β receptors. However, the Setd4Δ/Δ T-lymphomas were CD4+/CD8+ double positive, while the littermate Setd4flox/floxtumor were largely CD8+ single positive. A genomic sequencing analysis on chromosome deletion, inversion, duplication, and translocation, revealed a larger contribution of inversion but a less contribution of deletion to the overall chromosome rearrangements in the in Setd4Δ/Δ tumors than the Setd4flox/flox tumors. In addition, the Setd4flox/flox mice died more often from the large sizes of primary thymus lymphoma at earlier time, but there was a slight increase of lymphoma dissemination among peripheral organs in Setd4Δ/Δ at later times. These results suggest that Setd4 has a critical role in modulating lymphomagenesis and may be targeted to suppress radiation-induced carcinogenesis.

    更新日期:2019-11-26
  • Insights into the non-homologous end joining pathway and double strand break end mobility provided by mechanistic in silico modelling
    DNA Repair (IF 3.711) Pub Date : 2019-11-20
    John W. Warmenhoven, Nicholas T. Henthorn, Samuel P. Ingram, Amy L. Chadwick, Marios Sotiropoulos, Nickolay Korabel, Sergei Fedotov, Ranald I. Mackay, Karen J. Kirkby, Michael J. Merchant

    After radiation exposure, one of the critical processes for cellular survival is the repair of DNA double strand breaks. The pathways involved in this response are complex in nature and involve many individual steps that act across different time scales, all of which combine to produce an overall behaviour. It is therefore experimentally challenging to unambiguously determine the mechanisms involved and how they interact whilst maintaining strict control of all confounding variables. In silico methods can provide further insight into results produced by focused experimental investigations through testing of the hypotheses generated. Such computational testing can asses competing hypotheses by investigating their effects across all time scales concurrently, highlighting areas where further experimental work can have the most significance. We describe the construction of a mechanistic model by combination of several hypothesised mechanisms reported in the literature and supported by experiment. Compatibility of these mechanisms was tested by fitting simulation to results reported in the literature. To avoid over-fitting, we used an approach of sequentially testing individual mechanisms within this pathway. We demonstrate that using this approach the model is capable of reproducing published protein kinetics and overall repair trends. This provides evidence supporting the feasibility of the proposed mechanisms and revealed how they interact to produce an overall behaviour. Furthermore, we show that the assumed motion of individual double strand break ends plays a crucial role in determining overall system behaviour.

    更新日期:2019-11-21
  • Unhooking of an interstrand cross-link at DNA fork structures by the DNA glycosylase NEIL3
    DNA Repair (IF 3.711) Pub Date : 2019-11-20
    Maryam Imani Nejad, Kurt Housh, Alyssa A. Rodriguez, Tuhin Haldar, Scott Kathe, Susan S. Wallace, Brandt F. Eichman, Kent S. Gates
    更新日期:2019-11-21
  • Polymerase iota - an odd sibling among Y family polymerases
    DNA Repair (IF 3.711) Pub Date : 2019-11-20
    Justyna McIntyre

    It has been two decades since the discovery of the most mutagenic human DNA polymerase, polymerase iota (Polι). Since then, the biochemical activity of this translesion synthesis (TLS) enzyme has been extensively explored, mostly through in vitro experiments, with some insight into its cellular activity. Polι is one of four members of the Y-family of polymerases, which are the best characterized DNA damage-tolerant polymerases involved in TLS. Polι shares some common Y-family features, including low catalytic efficiency and processivity, high infidelity, the ability to bypass some DNA lesions, and a deficiency in 3′→5′ exonucleolytic proofreading. However, Polι exhibits numerous properties unique among the Y-family enzymes. Polι has an unusual catalytic pocket structure and prefers Hoogsteen over Watson-Crick pairing, and its replication fidelity strongly depends on the template; further, it prefers Mn2+ ions rather than Mg2+ as catalytic activators. In addition to its polymerase activity, Polι possesses also 5′-deoxyribose phosphate (dRP) lyase activity, and its ability to participate in base excision repair has been shown. As a highly error-prone polymerase, its regulation is crucial and mostly involves posttranslational modifications and protein-protein interactions. The upregulation and downregulation of Polι are correlated with different types of cancer and suggestions regarding the possible function of this polymerase have emerged from studies of various cancer lines. Nonetheless, after twenty years of research, the biological function of Polι certainly remains unresolved.

    更新日期:2019-11-21
  • Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination
    DNA Repair (IF 3.711) Pub Date : 2019-11-12
    Brian Ruis, Amy Molan, Taylor Takasugi, Eric A. Hendrickson

    The repair of DNA double-stranded breaks (DSBs) is an essential function performed by the Classical Non-Homologous End-Joining (C-NHEJ) pathway in higher eukaryotes. C-NHEJ, in fact, does double duty as it is also required for the repair of the intermediates formed during lymphoid B- and T-cell recombination. Consequently, the failure to properly repair DSBs leads to both genomic instability and immunodeficiency. A critical DSB protein required for C-NHEJ is the DNA Ligase IV (LIGIV) accessory factor, X-Ray Cross Complementing 4 (XRCC4). XRCC4 is believed to stabilize LIGIV, participate in LIGIV activation, and to help tether the broken DSB ends together. XRCC4′s role in these processes has been muddied by the identification of two additional XRCC4 paralogs, XRCC4-Like Factor (XLF), and Paralog of XRCC4 and XLF (PAXX). The roles that these paralogs play in C-NHEJ is partially understood, but, in turn, has itself been obscured by species-specific differences observed in the absence of one or the other paralogs. In order to investigate the role(s) that XRCC4 may play, with or without XLF and/or PAXX, in lymphoid variable(diversity)joining [V(D)J] recombination as well as in DNA DSB repair in human somatic cells, we utilized gene targeting to inactivate the XRCC4 gene in both parental and XLF− HCT116 cells and then inactivated PAXX in those same cell lines. The loss of XRCC4 expression by itself led, as anticipated, to increased sensitivity to DNA damaging agents as well as an increased dependence on microhomology-mediated DNA repair whether in the context of DSB repair or during V(D)J recombination. The additional loss of XLF in these cell lines sensitized the cells even more whereas the presence or absence of PAXX was scarcely negligible. These studies demonstrate that, of the three LIG4 accessory factor paralogs, the absence of XRCC4 influences DNA repair and recombination the most in human cells.

    更新日期:2019-11-18
  • Entrenching role of cell cycle checkpoints and autophagy for maintenance of genomic integrity
    DNA Repair (IF 3.711) Pub Date : 2019-11-13
    Sumit Kr Anand, Ankita Sharma, Neha Singh, Poonam Kakkar

    Genomic integrity of the cell is crucial for the successful transmission of genetic information to the offspring and its survival. Persistent DNA damage induced by endogenous and exogenous agents leads to various metabolic manifestations. To combat this, eukaryotes have developed complex DNA damage response (DDR) pathway which senses the DNA damage and activates an arsenal of enzymes for the repair of damaged DNA. The active pathways for DNA repair are nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR) for single-strand break repair whereas homologous recombination (HR) and non-homologous end-joining for double-strand break repair (NHEJ). OGG1 is a DNA glycosylase which initiates BER while Mre11-Rad50-Nbs1 (MRN) protein complex is the primary responder to DSBs which gets localized to damage sites. DNA damage response is meticulously executed by three related kinases: ATM, ATR, and DNA-PK. ATM- and ATR-dependent phosphorylation of p53, Chk1, and Chk2 regulate the G1/S, intra-S, or G2/M checkpoints of the cell cycle, respectively. Autophagy is an evolutionarily conserved process that plays a pivotal role in the regulation of DNA repair and maintains the cellular homeostasis. Genotoxic stress-induced altered autophagy occurs in a P53 dependent manner which is also the master regulator of genotoxic stress. A plethora of proteins involved in autophagy is regulated by p53 which involve DRAM, DAPK, and AMPK. As evident, the mtDNA is more prone to damage than nuclear DNA because of its close proximity to the site of ROS generation. Depending on the extent of damage either the repair mechanism or mitophagy gets triggered. SIRT1 is the master regulator which directs the stress response to mitophagy. Nix, a LC3 adapter also participates in Parkin mediated mitophagy. This review highlights the intricate crosstalks between DNA damage and cell cycle checkpoints activation. The DNA damage mediated regulation of autophagy and mitophagy is also reviewed in detail.

    更新日期:2019-11-18
  • RNases H: Structure and mechanism
    DNA Repair (IF 3.711) Pub Date : 2019-07-20
    Malwina Hyjek, Małgorzata Figiel, Marcin Nowotny

    RNases H are a family of endonucleases that hydrolyze RNA residues in various nucleic acids. These enzymes are present in all branches of life, and their counterpart domains are also found in reverse transcriptases (RTs) from retroviruses and retroelements. RNases H are divided into two main classes (RNases H1 and H2 or type 1 and type 2 enzymes) with common structural features of the catalytic domain but different range of substrates for enzymatic cleavage. Additionally, a third class is found in some Archaea and bacteria. Besides distinct cellular functions specific for each type of RNases H, this family of proteins is generally involved in the maintenance of genome stability with overlapping and cooperative role in removal of R-loops thus preventing their accumulation. Extensive biochemical and structural studies of RNases H provided not only a comprehensive and complete picture of their mechanism but also revealed key basic principles of nucleic acid recognition and processing. RNase H1 is present in prokaryotes and eukaryotes and cleaves RNA in RNA/DNA hybrids. Its main function is hybrid removal, notably in the context of R-loops. RNase H2, which is also present in all branches of life, can play a similar role but it also has a specialized function in the cleavage of single ribonucleotides embedded in the DNA. RNase H3 is present in Archaea and bacteria and is closely related to RNase H2 in sequence and structure but has RNase H1-like biochemical properties. This review summarizes the mechanisms of substrate recognition and enzymatic cleavage by different classes of RNases H with particular insights into structural features of nucleic acid binding, specificity towards RNA and/or DNA strands and catalysis.

    更新日期:2019-11-18
  • Drugging the R-loop interactome: RNA-DNA hybrid binding proteins as targets for cancer therapy
    DNA Repair (IF 3.711) Pub Date : 2019-07-06
    Beáta Boros-Oláh, Nikoletta Dobos, Lilla Hornyák, Zoltán Szabó, Zsolt Karányi, Gábor Halmos, Jason Roszik, Lóránt Székvölgyi

    Unravelling the origin of genetic alterations from point mutations to chromosomal rearrangements was greatly enhanced by the discovery of RNA-DNA hybrids (R-loops) that behave as hotspots of genomic instability in a variety of organisms. Current models suggest that uncontrolled R-loops are a hazard to genome integrity, therefore, identifying proteins that are involved in recognising and signalling R-loop structures are of key importance. Herein we analysed key RNA-DNA hybrid binding proteins in humans taking advantage of large-scale gene expression, survival rate, and drug-sensitivity data from cancer genomics databases. We show that expression of RNA-DNA hybrid binding proteins in various cancer types is associated with survival and may have contrasting outcomes in responding to therapeutic treatments. Based on the revealed pharmacogenomic landscape of human RNA-DNA hybrid binding proteins, we propose that R-loops and R-loop binding proteins are potentially relevant new epigenetic markers and therapeutic targets in multiple cancers.

    更新日期:2019-11-18
  • Genome-wide mutagenesis resulting from topoisomerase 1-processing of unrepaired ribonucleotides in DNA
    DNA Repair (IF 3.711) Pub Date : 2019-07-03
    Jessica S. Williams, Scott A. Lujan, Zhi-Xiong Zhou, Adam B. Burkholder, Alan B. Clark, David C. Fargo, Thomas A. Kunkel

    Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2–5 base pair deletions (Δ2–5bp) in repetitive DNA are a signature of unrepaired ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol ε, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.

    更新日期:2019-11-18
  • Pif1 family DNA helicases: A helpmate to RNase H?
    DNA Repair (IF 3.711) Pub Date : 2019-06-17
    Thomas J. Pohl, Virginia A. Zakian

    An R-loop is a structure that forms when an RNA transcript stays bound to the DNA strand that encodes it and leaves the complementary strand exposed as a loop of single stranded DNA. R-loops accumulate when the processing of RNA transcripts is impaired. The failure to remove these RNA-DNA hybrids can lead to replication fork stalling and genome instability. Resolution of R-loops is thought to be mediated mainly by RNase H enzymes through the removal and degradation of the RNA in the hybrid. However, DNA helicases can also dismantle R-loops by displacing the bound RNA. In particular, the Pif1 family DNA helicases have been shown to regulate R-loop formation at specific genomic loci, such as tRNA genes and centromeres. Here we review the roles of Pif1 family helicases in vivo and in vitro and discuss evidence that Pif1 family helicases act on RNA-DNA hybrids and highlight their potential roles in complementing RNase H for R-loop resolution.

    更新日期:2019-11-18
  • The Jekyll and Hyde character of RNase H1 and its multiple roles in mitochondrial DNA metabolism
    DNA Repair (IF 3.711) Pub Date : 2019-06-04
    Ian J. Holt

    The activity and specificity of ribonuclease H1, RNase H1, has been known for over half a century; like all enzymes in its class, it degrades RNA only when it is hybridized to DNA. However, the essential role of RNase H1 in mitochondrial DNA maintenance was not recognized until 2003, and empirical evidence that it is required to process RNA primers of mitochondrial DNA had to wait until 2015. In the same year, mutations in the RNASEH1 gene were linked to human mitochondrial diseases. The most recent studies suggest that in addition to primer-processing, RNase H1 determines the fate of R-loops, although not primarily those that might present an obstacle to DNA replication, but ones that contribute to the organization of mitochondrial DNA and the unusual mechanism of replication in mitochondria that utilizes transcripts for the strand-asynchronous mechanism of mitochondrial DNA replication. A full understanding of the role of RNase H1 in mtDNA metabolism will depend on further study, including careful consideration of its ability to stabilize, as well as to degrade RNA/DNA hybrids, and its regulation by oxidation or other mechanisms. Nevertheless, RNase H1 is already staking a strong claim to be the most versatile factor involved in propagating the DNA in the mitochondria.

    更新日期:2019-11-18
  • Genome instability consequences of RNase H2 Aicardi-Goutières syndrome alleles
    DNA Repair (IF 3.711) Pub Date : 2019-04-04
    Catherine J. Potenski, Anastasiya Epshtein, Christopher Bianco, Hannah L. Klein

    The RNase H2 complex is a conserved heterotrimeric enzyme that degrades RNA:DNA hybrids and promotes excision of rNMPs misincorporated during DNA replication. Failure to remove ribonucleotides from DNA leads to genomic instability in yeast and humans. The monogenic Aicardi-Goutières syndrome (AGS) results from mutation in one of several genes, among which are those encoding the RNase H2 subunits. The complete cellular and genomic consequences of RNASEH2 mutations and the precise connection to disease remain unclear. To learn more about the effect of RNASEH2 mutations on the cell, we used yeast as a model of AGS disease. We have generated yeast strains bearing AGS-associated mutations in RNASEH2 genes. There is a range of disease presentation in patients bearing these RNASEH2 variants. Here we report on in vivo phenotypes of genomic instability, including mutation and recombination rates, and synthetic gene interactions. These phenotypes provide insight into molecular consequences of RNASEH2 mutations, and lay the groundwork for further study of genomic instability as a contributing factor to AGS disease.

    更新日期:2019-11-18
  • Evaluation of DNA repair efficiency in autistic children by molecular cytogenetic analysis and transcriptome profiling
    DNA Repair (IF 3.711) Pub Date : 2019-11-14
    SM. Attia, MA. Al-Hamamah, SF. Ahmad, A. Nadeem, MSM. Attia, MA. Ansari, SA. Bakheet, LY. Al-Ayadhi

    Data regarding DNA repair perturbations in autism, which might increase the risk of malignancy, are scarce. To evaluate whether DNA repair may be disrupted in autistic children, we assessed the incidence of endogenous basal DNA strand breaks as well as the efficiency of repairing DNA damage caused by γ-ray in lymphocytes isolated from autistic and healthy children. The incidence of DNA damage and the kinetics of DNA repair were determined by comet assay, while the incidence of residual DNA damage was evaluated by structural chromosomal aberration analysis. Transcriptome profiling of 84 genes associated with DNA damage and repair-signaling pathways was performed by RT² Profiler PCR Array. The array data were confirmed by RT-PCR and western blot studies. Our data indicate that the incidence of basal oxidative DNA strand breaks in autistic children was greater than that in nonautistic controls. Lymphocytes from autistic children displayed higher susceptibility to damage by γ-irradiation and slower repair rate than those from nonautistic children. Although the total unstable chromosomal aberrations were unaffected, lymphocytes from autistic children were more susceptible to chromosomal damage caused by γ-ray than those from nonautistic children. Transcriptomic analysis revealed that several genes associated with repair were downregulated in lymphocytes from autistic individuals and in those exposed to γ-irradiation. This may explain the increased oxidative DNA damage and reduced repair rate in lymphocytes from autistic individuals. These features may be related to the possible correlation between autism and the elevated risk of cancer and may explain the role of the disruption of the DNA repair process in the pathogenesis of autism.

    更新日期:2019-11-14
  • Shining Light on the Response to Repair Intermediates in DNA of Living Cells
    DNA Repair (IF 3.711) Pub Date : 2019-11-12
    Agnes K. Janoshazi, Julie K. Horton, Ming-Lang Zhao, Rajendra Prasad, Erica L. Scappini, C. Jeff Tucker, Samuel H. Wilson

    Fluorescently-tagged repair proteins have been widely used to probe recruitment to micro-irradiation-induced nuclear DNA damage in living cells. Here, we quantify APE1 dynamics after micro-irradiation. Markers of DNA damage are characterized and UV-A laser micro-irradiation energy conditions are selected for formation of oxidatively-induced DNA base damage and single strand breaks, but without detectable double strand breaks. Increased energy of laser micro-irradiation, compared with that used previously in our work, enables study of APE1 dynamics at the lesion site. APE1 shows rapid transient kinetics, with recruitment half-time of less than 1 s and dissociation half-time of less than 15 s. In cells co-transfected with APE1 and PARP1, the recruitment half-time of PARP1 was slower than that of APE1, indicating APE1 is a rapid responder to the damage site. While recruitment of APE1 is unchanged in the presence of co-transfected PARP1, APE1 dissociation is 3-fold slower, revealing PARP1 involvement in APE1 dynamics. Further, we find that APE1 dissociation kinetics are strongly modified in the absence of DNA polymerase β (pol β). After unchanged recruitment to the damage site, dissociation of APE1 became undetectable. This indicates a necessary role for pol β in APE1 release after its recruitment to the damage site. These observations represent an advance in our understanding of in vivo dynamics of base excision repair factors APE1, PARP1 and pol β.

    更新日期:2019-11-13
  • s Sp1-independent downregulation of NHEJ in response to BER deficiency
    DNA Repair (IF 3.711) Pub Date : 2019-11-11
    Polina S. Loshchenova, Svetlana V. Sergeeva, Dmitry V. Limonov, Zhigang Guo, Grigory L. Dianov

    Base excision repair (BER) is the major repair pathway that removes DNA single strand breaks (SSBs) arising spontaneously due to the inherent instability of DNA. Unrepaired SSBs promote cell-cycle delay, which facilitates DNA repair prior to replication. On the other hand, in response to persistent DNA strand breaks, ATM-dependent degradation of transcription factor Sp1 leads to downregulation of BER genes expression, further accumulation of SSBs and renders cells susceptible to elimination via apoptosis. In contrast, many cancer cells are not able to block replication and to downregulate the expression of Sp1 in response to DNA damage. However, knockdown of BER in cancer cells leads to the accumulation of DNA double strand breaks (DSBs), suggesting deficiency in non-homologous end joining (NHEJ) repair of DSBs. Here we investigated whether DNA repair deficiency caused by knockdown of the XRCC1 gene expression in proliferating cells results in downregulation of NHEJ genes expression. We find that knockdown of the XRCC1 gene expression does not cause degradation of Sp1, but leads to downregulation of Lig4/XRCC4 and Ku70/80 at the transcription and protein levels. We thus propose the existence of Sp1-independent backup mechanism that in response to BER deficiency downregulates NHEJ in proliferating cells.

    更新日期:2019-11-13
  • Deletion of the SAPS1 subunit of protein phosphatase 6 in mice increases radiosensitivity and impairs the cellular DNA damage response
    DNA Repair (IF 3.711) Pub Date : 2019-11-09
    Jaroslaw Dziegielewski, Magdalena A. Bońkowska, Ewa A. Poniecka, Jinho Heo, Kangping Du, Rowena B. Crittenden, Timothy P. Bender, David L. Brautigan, James M. Larner

    Cellular responses to DNA damage include activation of DNA-dependent protein kinase (DNA-PK) through, among others, the serine/threonine protein phosphatase 6 (PP6). We previously showed that recognition of DNA-PKcs is mediated by the SAPS1 PP6 regulatory subunit. Here, we report and characterize a SAPS1 null mouse and investigate the effects of deletion on DNA damage signaling and repair. Strikingly, neither SAPS1-null animals nor cells derived from them show gross defects, unless subjected to DNA damage by radiation or chemical agents. The overall survival of SAPS1-null animals following whole body irradiation is significantly shortened as compared to wild-type mice, and the clonogenic survival of null cells subjected to ionizing radiation is reduced. The dephosphorylation of DNA damage/repair markers, such as γH2AX, p53 and Kap1, is diminished in SAPS1-null cells as compared to wild-type controls. Our results demonstrate that loss of SAPS1 confers sensitivity to DNA damage and confirms previously reported cellular phenotypes of SAPS1 knock-down in human glioma cells. The results support a role for PP6 regulatory subunit SAPS1 in DNA damage responses, and offer a novel target for sensitization to enhance current tumor therapies, with a potential for limited deleterious side effects.

    更新日期:2019-11-11
  • A combined structural and biochemical approach reveals translocation and stalling of UvrB on the DNA lesion as a mechanism of damage verification in bacterial nucleotide excision repair
    DNA Repair (IF 3.711) Pub Date : 2019-11-06
    Marcin Jaciuk, Paolo Swuec, Vineet Gaur, Joanna M. Kasprzak, Ludovic Renault, Mateusz Dobrychłop, Shivlee Nirwal, Janusz M. Bujnicki, Alessandro Costa, Marcin Nowotny

    Nucleotide excision repair (NER) is a DNA repair pathway present in all domains of life. In bacteria, UvrA protein localizes the DNA lesion, followed by verification by UvrB helicase and excision by UvrC double nuclease. UvrA senses deformations and flexibility of the DNA duplex without precisely localizing the lesion in the damaged strand, an element essential for proper NER. Using a combination of techniques, we elucidate the mechanism of the damage verification step in bacterial NER. UvrA dimer recruits two UvrB molecules to its two sides. Each of the two UvrB molecules clamps a different DNA strand using its β-hairpin element. Both UvrB molecules then translocate to the lesion, and UvrA dissociates. The UvrB molecule that clamps the damaged strand gets stalled at the lesion to recruit UvrC. This mechanism allows UvrB to verify the DNA damage and identify its precise location triggering subsequent steps in the NER pathway.

    更新日期:2019-11-06
  • Novel Deazaflavin Tyrosyl-DNA Phosphodiesterase 2 (TDP2) Inhibitors
    DNA Repair (IF 3.711) Pub Date : 2019-11-06
    Evgeny Kiselev, Azhar Ravji, Jayakanth Kankanala, Jiashu Xie, Zhengqiang Wang, Yves Pommier

    Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a DNA repair enzyme that removes 5′-phosphotyrosyl blockages resulting from topoisomerase II (TOP2)-DNA cleavage complexes trapped by TOP2 inhibitors. TDP2 is a logical target for the development of therapeutics to complement existing treatments based on inhibition of TOP2. There is, however, no TDP2 inhibitor in clinical development at present. Of the reported TDP2 inhibitors, the deazaflavins are the most promising chemical class centered around the lead compound SV-5-153. Recently we reported new subtypes derived within the deazaflavin family with improved membrane permeability properties. In this work we characterize two representative analogues from two new deazaflavin subtypes based on their biochemical TDP2 inhibitory potency and drug-likeness. We demonstrate that the ZW-1288 derivative represents a promising direction for the development of deazaflavins as therapeutic agents. ZW-1288 exhibits potent inhibitory activity at low nanomolar concentrations against recombinant and cellular human TDP2 with profile similar to that of the parent analog SV-5-153 based on high resistance against murine TDP2 and human TDP2 mutated at residue L313H. While expressing weak cytotoxicity on its own, ZW-1288 potentiates the clinical TOP2 inhibitors etoposide (ETP) and mitoxantrone in human prostate DU145 and CCRF-CEM leukemia and chicken lymphoma DT40 cells while not impacting the activity of the topoisomerase I (TOP1) inhibitor camptothecin or the PARP inhibitor olaparib. ZW-1288 increases the uptake of ETP to a lesser extent than SV-5-153 and remained active in TDP2 knockout cells indicating that the deazaflavin TDP2 inhibitors have additional cellular effects that will have to be taken into account for their further development as TDP2 inhibitors.

    更新日期:2019-11-06
  • Integration Host Factor IHF facilitates homologous recombination and mutagenic processes in Pseudomonas putida
    DNA Repair (IF 3.711) Pub Date : 2019-11-05
    Katren Mikkel, Mari Tagel, Kärt Ukkivi, Heili Ilves, Maia Kivisaar

    Nucleoid-associated proteins (NAPs) such as IHF, HU, Fis, and H-NS alter the topology of bound DNA and may thereby affect accessibility of DNA to repair and recombination processes. To examine this possibility, we investigated the effect of IHF on the frequency of homologous recombination (HR) and point mutations in soil bacterium Pseudomonas putida by using plasmidial and chromosomal assays. We observed positive effect of IHF on the frequency of HR, whereas this effect varied depending both on the chromosomal location of the HR target and the type of plasmid used in the assay. The occurrence of point mutations in plasmid was also facilitated by IHF, whereas in the chromosome the positive effect of IHF appeared only at certain DNA sequences and/or chromosomal positions. We did not observe any significant effects of IHF on the spectrum of mutations. However, despite of the presence or absence of IHF, different mutational hot spots appeared both in plasmid and in chromosome. Additionally, the frequency of frameshift mutations in the chromosome was also strongly affected by the location of the mutational target sequence. Taking together, our results indicate that IHF facilitates the occurrence of genetic changes in P. putida, whereas the location of the target sequence affects both the IHF-dependent and IHF-independent mechanisms.

    更新日期:2019-11-06
  • Recognition of DNA adducts by edited and unedited forms of DNA glycosylase NEIL1
    DNA Repair (IF 3.711) Pub Date : 2019-11-02
    Irina G. Minko, Vladimir L. Vartanian, Naoto N. Tozaki, Erdem Coskun, Sanem Hosbas Coskun, Pawel Jaruga, Jongchan Yeo, Sheila S. David, Michael P. Stone, Martin Egli, Miral Dizdaroglu, Amanda K. McCullough, R. Stephen Lloyd

    Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ≈30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ≈3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, γ-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all the measured DNA lesions, imidazole ring-opened 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were preferentially released by both NEIL1 enzymes with K242R being ≈1.3 and 1.2-fold more efficient than K242 on excision of FapyAde and FapyGua, respectively. Consistent with the prior literature, large differences (≈7.5 to 12-fold) were measured in the excision of ThyGly from genomic DNA by the unedited versus edited NEIL1. In contrast, the edited NEIL1 was more efficient (≈3 to 5-fold) on release of 5-hydroxy-cytosine. Excision kinetics on DNA containing a site-specific aflatoxin B1-FapyGua adduct revealed an ≈1.4-fold higher rate by the unedited NEIL1. Molecular modeling provides insight into these differential substrate specificities. The results of this study and in particular, the comparison of substrate specificities of unedited and edited NEIL1 using biologically and clinically important base lesions, are critical for defining its role in preservation of genomic integrity.

    更新日期:2019-11-04
  • Cutting-edge perspectives in genomic maintenance VI.
    DNA Repair (IF 3.711) Pub Date : 2019-08-21
    Philip C Hanawalt,Samuel H Wilson

    更新日期:2019-11-01
  • A tribute in memory of Richard B. (Dick) Setlow (1921-2015).
    DNA Repair (IF 3.711) Pub Date : 2015-10-16
    Phil Hanawalt,Arthur Grollman,SankarMitra

    更新日期:2019-11-01
  • XRCC1 interaction with the REV1 C-terminal domain suggests a role in post replication repair.
    DNA Repair (IF 3.711) Pub Date : 2014-01-11
    Scott A Gabel,Eugene F DeRose,Robert E London

    The function of X-ray cross complementing group 1 protein (XRCC1), a scaffold that binds to DNA repair enzymes involved in single-strand break and base excision repair, requires that it be recruited to sites of damaged DNA. However, structural insights into this recruitment are currently limited. Sequence analysis of the first unstructured linker domain of XRCC1 identifies a segment consistent with a possible REV1 interacting region (X1RIR) motif. The X1RIR motif is present in translesion polymerases that can be recruited to the pol /REV1 DNA repair complex via a specific interaction with the REV1 C-terminal domain. NMR and fluorescence titration studies were performed on XRCC1-derived peptides containing this putative RIR motif in order to evaluate the binding affinity for the REV1 C-terminal domain. These studies demonstrate an interaction of the XRCC1-derived peptide with the human REV1 C-terminal domain characterized by dissociation constants in the low micromolar range. Ligand competition studies comparing the XRCC1 RIR peptide with previously studied RIR peptides were found to be inconsistent with the NMR based Kd values. These discrepancies were resolved using a fluorescence assay for which the RIR–REV1 system is particularly well suited. The structure of a REV1-XRCC1 peptide complex was determined by using NOE restraints to dock the unlabeled XRCC1 peptide with a labeled REV1 C-terminal domain. The structure is generally homologous with previously determined complexes with the pol κ and pol η RIR peptides, although the helical segment in XRCC1 is shorter than was observed in these cases. These studies suggest the possible involvement of XRCC1 and its associated repair factors in post replication repair.

    更新日期:2019-11-01
  • A life of fixing DNA.
    DNA Repair (IF 3.711) Pub Date : 2013-07-06
    Errol C Friedberg

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • My long and winding road to mutagenesis and DNA repair pathways.
    DNA Repair (IF 3.711) Pub Date : 2013-04-27
    Jeffrey H Miller

    更新日期:2019-11-01
  • Autobiographical sketch: A life in DNA repair—and beyond.
    DNA Repair (IF 3.711) Pub Date : 2012-12-06
    Hans E Krokan

    更新日期:2019-11-01
  • Losing and finding myself in DNA repair.
    DNA Repair (IF 3.711) Pub Date : 2012-09-27
    Larry H Thompson

    更新日期:2019-11-01
  • My path toward DNA repair.
    DNA Repair (IF 3.711) Pub Date : 2012-08-08
    Mutsuo Sekiguchi

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • An accidental biochemist.
    DNA Repair (IF 3.711) Pub Date : 2012-07-19
    Myron F Goodman

    更新日期:2019-11-01
  • Repairing DNA for 80 years: the timeline of my life.
    DNA Repair (IF 3.711) Pub Date : 2012-06-12
    Phil Hanawalt

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Successes and failures in early DNA repair research.
    DNA Repair (IF 3.711) Pub Date : 2011-12-23
    Robert Painter

    更新日期:2019-11-01
  • The recombination, repair and modification of DNA.
    DNA Repair (IF 3.711) Pub Date : 2011-11-09
    Robin Holliday

    This article is an overview of the author's involvement in theoretical and experimental research on genetic recombination and DNA repair, and also on the enzymic modification of cytosine in DNA to 5-methyl cytosine. It includes the history of the discovery of the central intermediate in genetic recombination at the DNA level, and the repair of mismatched bases. These explain the major features of genetic fine structure. The first repair and recombination defective mutants in any eukaryote were isolated in the smut fungus Ustilago maydis. The hypothesis that DNA methylation has a role in gene expression in higher organism is now supported by abundant evidence. Direct evidence that gene silencing in mammalian cells is causally related to DNA methylation has been obtained.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • 更新日期:2019-11-01
  • The Drosophila mus 308 gene product, implicated in tolerance of DNA interstrand crosslinks, is a nuclear protein found in both ovaries and embryos.
    DNA Repair (IF 3.711) Pub Date : 2005-06-18
    Maria Pang,Maeve McConnell,Paul A Fisher

    mus 308 designates one of over 30 mutagen sensitivity loci found in Drosophila. It is predicted to code for a 229-kDa polypeptide. Published sequence analyses of others indicate that this polypeptide would have helicase motifs near its N-terminus, and similarities to bacterial DNA polymerase I-like enzymes near its C-terminus. In our studies, two different and highly specific antibodies were prepared and used for identification as well as characterization of the mus 308 gene product. Western blot analyses reveal a single reactive polypeptide in both ovaries and embryos as well as in two Drosophila embryo tissue culture cell lines; it is nearly absent in homozygous mus 308 mutants. This polypeptide is about 229 kDa in size, and indirect immunofluorescence shows that the mus 308 gene product localizes throughout nuclei in wild-type cells but appears to be absent in a mus 308 mutant. Immunoblot analyses throughout development suggest greatest abundance at the end of embryogenesis, immediately before hatching of first instar larvae. They also showed a smaller ( approximately 100 kDa) antigenically and genetically related polypeptide found only in adult males. Immunoprecipitation, a highly effective method of specific purification, suggests that the mus 308 protein has DNA polymerase activity that is NEM-sensitive but largely aphidicolin-resistant. In addition, the immunoprecipitated material has DNA-dependent ATPase but lacks detectable helicase.

    更新日期:2019-11-01
  • RNases H: Multiple roles in maintaining genome integrity.
    DNA Repair (IF 3.711) Pub Date : null
    Susana M Cerritelli,Robert J Crouch

    更新日期:2019-11-01
  • Obituary: Arthur Kornberg (1918-2007).
    DNA Repair (IF 3.711) Pub Date : 2008-04-01
    Errol C Friedberg

    更新日期:2019-11-01
  • "Meeting Report DNA Repair 2006: Ninth Biennial Meeting of the German Society for Research on DNA Repair" [DNA Repair 6 (2007) 140-144].
    DNA Repair (IF 3.711) Pub Date : 2007-06-30
    Jochen Dahm-Daphi,Lisa Wiesmüller,Ekkehard Dikomey

    A comprehensive meeting on current aspects of DNA repair organized by, Jochen Dahm-Daphi, Ekkehard Dikomey, Alexander Bürkle, Marlis Frankenberg-Schwager, Frank Grosse, Andrea Hartwig, George Iliakis, Bernd Kaina, Jürgen Thomale, and Lisa Wiesmüller was held in Hamburg, Germany from 12 to 15 September 2006. This article summarizes information of invited lectures and proferred papers of six plenary sessions.

    更新日期:2019-11-01
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