| 1. |
- Elmroth, Kerstin, 1970, et al.
(author)
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Cleavage of cellular DNA by calicheamicin γ1
- 2003
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In: DNA Repair. - 1568-7864 .- 1568-7856. ; 2:4, s. 363-374
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Journal article (peer-reviewed)abstract
- It is assumed that the efficient antitumor activity of calicheamicin γ1 is mediated by its ability to introduce DNA double-strand breaks in cellular DNA. To test this assumption we have compared calicheamicin γ1-mediated cleavage of cellular DNA and purified plasmid DNA. Cleavage of purified plasmid DNA was not inhibited by excess tRNA or protein indicating that calicheamicin γ1 specifically targets DNA. Cleavage of plasmid DNA was not affected by incubation temperature. In contrast, cleavage of cellular DNA was 45-fold less efficient at 0°C as compared to 37° due to poor cell permeability at low temperatures. The ratio of DNA double-strand breaks (DSB) to single-stranded breaks (SSB) in cellular DNA was 1:3, close to the 1:2 ratio observed when calicheamicin γ1 cleaved purified plasmid DNA. DNA strand breaks introduced by calicheamicin γ1 were evenly distributed in the cell population as measured by the comet assay. Calicheamicin γ1-induced DSBs were repaired slowly but completely and resulted in high levels of H2AX phosphorylation and efficient cell cycle arrest. In addition, the DSB-repair deficient cell line Mo59J was hyper sensitive to calicheamicin γ. The data indicate that DSBs is the crucial damage after calicheamicin γ1 and that calicheamicin γ1-induced DSBs are recognized normally. The high DSB:SSB ratio, specificity for DNA and the even damage distribution makes calicheamicin γ1 a superior drug for studies of the DSB-response and emphasizes its usefulness in treatment of malignant disease.
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| 2. |
- Singh, Vandana, 1985, et al.
(author)
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Shining light on single-strand lesions caused by the chemotherapy drug bleomycin
- 2021
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In: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 105
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Journal article (peer-reviewed)abstract
- Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses.
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| 3. |
- Keane, Simon, et al.
(author)
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DLG2 impairs dsDNA break repair and maintains genome integrity in neuroblastoma
- 2022
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In: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 112
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Journal article (peer-reviewed)abstract
- BackgroundIn primary neuroblastoma, deletions on chromosome 11q are known to result in an increase in the total number of chromosomal breaks. The DNA double-strand break repair pathways mediated by NHEJ are often upregulated in cancer. DLG2, a candidate tumor suppressor gene on chromosome 11q, has previously been implicated in DNA repair.MethodsWe evaluated an association between gene expression and neuroblastoma patient outcome, risk categorization, and 11q status using publicly available microarray data from independent neuroblastoma patient datasets. Functional studies were conducted using comet assay and H2AX phosphorylation in neuroblastoma cell lines and in the fruit fly with UVC-induced DNA breaks.ResultsWe show that the NHEJ genes PARP1 and FEN1 are over expressed in neuroblastoma and restoration of DLG2 impairs their gene and protein expression. When exposed to UVC radiation, cells with DLG2 over expression show less DNA fragmentation and induce apoptosis in a p53 S46 dependent manner. We could also confirm that DLG2 over expression results in CHK1 phosphorylation consistent with previous reports of G2/M maintenance.ConclusionsTaken together, we show that DLG2 over expression increases p53 mediated apoptosis in response to etoposide and UVC mediated genotoxicity and reduced DNA replication machinery.
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| 4. |
- Stone, Jana E, et al.
(author)
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Lesion bypass by S. cerevisiae Pol ζ alone
- 2011
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In: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 10:8, s. 826-834
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Journal article (peer-reviewed)abstract
- DNA polymerase zeta (Pol ζ) participates in translesion synthesis (TLS) of DNA adducts that stall replication fork progression. Previous studies have led to the suggestion that the primary role of Pol ζ in TLS is to extend primers created when another DNA polymerase inserts nucleotides opposite lesions. Here we test the non-exclusive possibility that Pol ζ can sometimes perform TLS in the absence of any other polymerase. To do so, we quantified the efficiency with which S. cerevisiae Pol ζ bypasses abasic sites, cis-syn cyclobutane pyrimidine dimers and (6-4) photoproducts. In reactions containing dNTP concentrations that mimic those induced by DNA damage, a Pol ζ derivative with phenylalanine substituted for leucine 979 at the polymerase active site bypasses all three lesions at efficiencies between 27 and 73%. Wild-type Pol ζ also bypasses these lesions, with efficiencies that are lower and depend on the sequence context in which the lesion resides. The results are consistent with the hypothesis that, in addition to extending aberrant termini created by other DNA polymerases, Pol ζ has the potential to be the sole DNA polymerase involved in TLS.
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| 5. |
- Watt, Danielle L, et al.
(author)
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Replication of ribonucleotide-containing DNA templates by yeast replicative polymerases
- 2011
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In: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 10:8, s. 897-902
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Journal article (peer-reviewed)abstract
- The major replicative DNA polymerases of S. cerevisiae (Pols α, δ, and ɛ) incorporate substantial numbers of ribonucleotides into DNA during DNA synthesis. When these ribonucleotides are not removed in vivo, they reside in the template strand used for the next round of replication and could potentially reduce replication efficiency and fidelity. To examine if the presence of ribonucleotides in a DNA template impede DNA synthesis, we determined the efficiency with which Pols α, δ, and ɛ copy DNA templates containing a single ribonucleotide. All three polymerases can replicate past ribonucleotides. Relative to all-DNA templates, bypass of ribo-containing templates is slightly reduced, to extents that depend on the identity of the ribo and the sequence context in which it resides. Bypass efficiencies for Pols δ and ɛ were increased by increasing the dNTP concentrations to those induced by cellular stress, and in the case of Pol ɛ, by inactivating the 3'-exonuclease activity. Overall, ribonucleotide bypass efficiencies are comparable to, and usually exceed, those for the common oxidative stress-induced lesion 8-oxo-guanine.
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| 6. |
- Wollen Steen, Kristian, et al.
(author)
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MtSSB may sequester UNG1 at mitochondrial ssDNA and delay uracil processing until the dsDNA conformation is restored
- 2012
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In: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 11:1, s. 82-91
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Journal article (peer-reviewed)abstract
- Single-strand DNA binding proteins protect DNA from nucleolytic damage, prevent formation of secondary structures and prevent premature reannealing of DNA in DNA metabolic transactions. In eukaryotes, the nuclear single-strand DNA binding protein RPA is essential for chromosomal DNA replication and transcription and directly participates in several DNA repair processes by binding to and modulating the activity of repair factors. Much less is known about the involvement of the only mitochondrial single-strand binding protein mtSSB in the context of DNA repair. Here we demonstrate that mtSSB impedes excision of uracil and oxidative demethylation of 3meC in single-stranded DNA by UNG1 and ABH1, respectively, whereas excision by NEIL1 was partially inhibited. mtSSB also effectively inhibited nicking of single-stranded DNA by APE1 and ABH1 and partially inhibited the lyase activity of NEIL1. Finally we identified a putative surface motif in mtSSB that may recruit UNG1 to DNA-bound mtSSB. We suggest that the massive amount of mtSSB in mitochondria effectively prevents processing of uracil and other types of damaged bases to avoid introduction of nicks in single-stranded mtDNA formed during replication. Local enrichment of UNG1 at DNA-bound mtSSB may furthermore facilitate rapid access to- and processing of the damage once the dsDNA conformation is restored. This could be of potential biological importance, since mitochondria have no or limited capacity for homologous recombination to process nicks at the replication fork. © 2011 Elsevier B.V.
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| 7. |
- Farnebo, Lovisa, et al.
(author)
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DNA repair genes XPC, XPD, XRCC1, and XRCC3 are associated with risk and survival of squamous cell carcinoma of the head and neck
- 2015
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In: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 31, s. 64-72
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Journal article (peer-reviewed)abstract
- Head and neck squamous cell carcinomas (HNSCC) are a heterogenous group of tumors with a high rate of early recurrences, second primary tumors, and mortality. Despite advances in diagnosis and treatment over the past decades, the overall 5-year survival rate remains around 50%. Since the head-and neck-region is continuously exposed to potentially DNA-damaging exogenous and endogenous factors, it is reasonable to expect that the DNA repair genes play a part in the development, progression, and outcome of HNSCC. The aim of this study was to investigate the SNPs XPC A499V, XPD K751Q XRCC1 R399Q and XRCC3 T241M as potential risk factors and indicators of survival among Caucasian patients. One-hundred-sixty-nine patients as well as 344 healthy controls were included and genotyped with PCR-RFLP. We showed that XPC A499V was associated with increased risk of HNSCC, especially laryngeal carcinoma. Among women, XPD K751Q was associated with increased risk of oral SCC. Furthermore, XPD homozygous mutant individuals had the shortest survival time, a survival time that increased however after full dose radiotherapy. Wild-type individuals of XRCC3 T241M demonstrated an earlier age of onset. HPV-positive never smokers had lower frequencies of p53 mutation. Among HNSCC patients, HPV-positivity was significantly associated with XRCC1 R399Q homozygous mutant genotype. Moreover, combinations of putative risk alleles seemed to act synergistically, increasing the risk of HNSCC. In conclusion, our results suggest that SNPs of the DNA repair genes XPC, XPD, XRCC1, and XRCC3 may affect risk and survival of HNSCC. (C) 2015 Elsevier B.V. All rights reserved.
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| 8. |
- Akuwudike, Pamela, 1987-, et al.
(author)
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Mechanistic insights from high resolution DNA damage analysis to understand mixed radiation exposure
- 2023
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In: DNA Repair. - 1568-7864 .- 1568-7856. ; 130
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Journal article (peer-reviewed)abstract
- Cells exposed to densely ionising high and scattered low linear energy transfer (LET) radiation (50 % dose of each) react more strongly than to the same dose of each separately. The relationship between DNA double strand break location inside the nucleus and chromatin structure was evaluated, using high-resolution transmission electron microscopy (TEM) in breast cancer MDA-MB-231 cells at 30 min post 5 Gy. Additionally, response to high and/or low LET radiation was assessed using single (1 ×1.5 Gy) versus fractionated dose delivery (5 ×0.3 Gy). By TEM analysis, the highest total number of γH2AX nanobeads were found in cells irradiated with alpha radiation just prior to gamma radiation (called mixed beam), followed by alpha, then gamma radiation. γH2AX foci induced by mixed beam radiation tended to be surrounded by open chromatin (lighter TEM regions), yet foci containing the highest number of beads, i.e. larger foci representing complex damage, remained in the heterochromatic areas. The γH2AX large focus area was also greater in mixed beam-treated cells when analysed by immunofluorescence. Fractionated mixed beams given daily induced the strongest reduction in cell viability and colony formation in MDA-MB-231 and osteosarcoma U2OS cells compared to the other radiation qualities, as well as versus acute exposure. This may partially be explained by recurring low LET oxidative DNA damage by every fraction together with a delay in recompaction of chromatin after high LET, demonstrated by low levels of heterochromatin marker H3K9me3 at 2 h after the last mixed beam fraction in MDA-MB-231. In conclusion, early differences in response to complex DNA damage may lead to a stronger cell kill induced by fractionated exposure, which suggest a therapeutic potential of combined high and low LET irradiation.
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| 9. |
- Andersen, Sonja, et al.
(author)
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Monoclonal B-cell hyperplasia and leukocyte imbalance precede development of B-cell malignancies in uracil-DNA glycosylase deficient mice
- 2005
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In: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 4:12, s. 1432-1441
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Journal article (peer-reviewed)abstract
- Ung-deficient mice have reduced class switch recombination, skewed somatic hypermutation, lymphatic hyperplasia and a 22-fold increased risk of developing B-cell lymphomas. We find that lymphomas are of follicular (FL) and diffuse large B-cell type (DLBCL). All FLs and 75% of the DLBCLs were monoclonal while 25% were biclonal. Monoclonality was also observed in hyperplasia, and could represent an early stage of lymphoma development. Lymphoid hyperplasia occurs very early in otherwise healthy Ung-deficient mice, observed as a significant increase of splenic B-cells. Furthermore, loss of Ung also causes a significant reduction of T-helper cells, and 50% of the young Ung(-/-) mice investigated have no detectable NK/NKT-cell population in their spleen. The immunological imbalance is confirmed in experiments with spleen cells where the production of the cytokines interferon gamma, interleukin 6 and interleukin 2 is clearly different in wild type and in Ung-deficient mice. This suggests that Ung-proteins, directly or indirectly, have important functions in the immune system, not only in the process of antibody maturation, but also for production and functions of immunologically important cell types. The immunological imbalances shown here in the Ung-deficient mice may be central in the development of lymphomas in a background of generalised lymphoid hyperplasia.
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| 10. |
- Bochman, Matthew L, et al.
(author)
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Unwinding the functions of the Pif1 family helicases
- 2010
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In: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 9:3, s. 237-249
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Journal article (peer-reviewed)abstract
- Helicases are ubiquitous enzymes found in all organisms that are necessary for all (or virtually all) aspects of nucleic acid metabolism. The Pif1 helicase family is a group of 5'-->3' directed, ATP-dependent, super family IB helicases found in nearly all eukaryotes. Here, we review the discovery, evolution, and what is currently known about these enzymes in Saccharomyces cerevisiae (ScPif1 and ScRrm3), Schizosaccharomyces pombe (SpPfh1), Trypanosoma brucei (TbPIF1, 2, 5, and 8), mice (mPif1), and humans (hPif1). Pif1 helicases variously affect telomeric, ribosomal, and mitochondrial DNA replication, as well as Okazaki fragment maturation, and in at least some cases affect these processes by using their helicase activity to disrupt stable nucleoprotein complexes. While the functions of these enzymes vary within and between organisms, it is evident that Pif1 family helicases are crucial for both nuclear and mitochondrial genome maintenance.
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