http://www.genomeintegrity.com The latest research articles published by Genome Integrity 2012-01-26T00:00:00Z Background: The quantification of radiation-induced foci (RIF) to investigate the induction and subsequent repair of DNA double strands breaks is now commonplace. Over the last decade systems specific for the automatic quantification of RIF have been developed for this purpose, however to ask more mechanistic questions on the spatio-temporal aspects of RIF, an automated RIF analysis platform that also quantifies RIF size/volume and relative three-dimensional (3D) distribution of RIF within individual nuclei, is required. Results: A java-based image analysis system has been developed (AutoRIF) that quantifies the number, size/volume and relative nuclear locations of RIF within 3D nuclear volumes. Our approach identifies nuclei using the dynamic Otsu threshold and RIF by enhanced Laplacian filtering and maximum entropy thresholding steps and, has an application 'batch optimisation' process to ensure reproducible quantification of RIF. AutoRIF was validated by comparing output against manual quantification of the same 2D and 3D image stacks with results showing excellent concordance over a whole range of sample time points (and therefore range of total RIF/nucleus) after low-LET radiation exposure. Conclusions: This high-throughput automated RIF analysis system generates data with greater depth of information and reproducibility than that which can be achieved manually and may contribute toward the standardisation of RIF analysis. In particular, AutoRIF is a powerful tool for studying spatio-temporal relationships of RIF using a range of DNA damage response markers and can be run independently of other software, enabling most personal computers to perform image analysis. Future considerations for AutoRIF will likely include more complex algorithms that enable multiplex analysis for increasing combinations of cellular markers. http://www.genomeintegrity.com/content/3/1/1 Andrew McVean Simon Kent Alexei Bakanov Tom Hobbs Rhona Anderson Genome Integrity 2012, null:1 2012-01-26T00:00:00Z doi:10.1186/2041-9414-3-1 /content/figures/2041-9414-3-1-toc.gif Genome Integrity 2041-9414 ${item.volume} 1 2012-01-26T00:00:00Z PDF Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. http://www.genomeintegrity.com/content/2/1/10 Tyson Clark Kristi Spittle Stephen Turner Jonas Korlach Genome Integrity 2011, null:10 2011-12-20T00:00:00Z doi:10.1186/2041-9414-2-10 /content/figures/2041-9414-2-10-toc.gif Genome Integrity 2041-9414 ${item.volume} 10 2011-12-20T00:00:00Z XML Background: Recent studies suggest that BRCA2 affects telomere maintenance. Interestingly, anti cancer treatments that involve BRCA2 and telomerase individually are currently being explored. In the light of the above recent studies their combinatorial targeting may be justified in the development of future treatments. In order to investigate effects of BRCA2 that can be explored for this combinatorial targeting we focused on the analysis of recombination rates at telomeres by monitoring T-SCEs (Telomere Sister Chromatid Exchanges). Results: We observed a significant increase in T-SCE frequencies in four BRCA2 defective human cell lines thus suggesting that BRCA2 suppresses recombination at telomeres. To test this hypothesis further we analyzed T-SCE frequencies in a set of Chinese hamster cell lines with or without functional BRCA2. Our results indicate that introduction of functional BRCA2 normalizes frequencies of T-SCEs thus supporting the notion that BRCA2 suppresses recombination at telomeres. Given that ALT (Alternative Lengthening of Telomeres) positive cells maintain telomeres by recombination we investigated the effect of BRCA2 depletion in these cells. Our results show that this depletion causes a dramatic reduction in T-SCE frequencies in ALT positive cells, but not in non-ALT cells. Conclusion: BRCA2 suppresses recombination at telomeres in cells that maintain them by conventional mechanisms. Furthermore, BRCA2 depletion in ALT positive cells reduces high levels of T-SCEs normally found in these cells. Our results could be potentially important for refining telomerase-based anti-cancer therapies. http://www.genomeintegrity.com/content/2/1/9 Ester Sapir Yaghoub Gozaly-Chianea Suliman Al-Wahiby Sainu Ravindran Hemad Yasaei Predrag Slijepcevic Genome Integrity 2011, null:9 2011-12-09T00:00:00Z doi:10.1186/2041-9414-2-9 /content/figures/2041-9414-2-9-toc.gif Genome Integrity 2041-9414 ${item.volume} 9 2011-12-09T00:00:00Z XML Background: Genome instability is associated with human cancers and chromosome breakage syndromes, including Bloom's syndrome, caused by inactivation of BLM helicase. Numerous mutations that lead to genome instability are known, yet how they interact genetically is poorly understood. Results: We show that spontaneous translocations that arise by nonallelic homologous recombination in DNA-damage-checkpoint-defective yeast lacking the BLM-related Sgs1 helicase (sgs1Δ mec3Δ) are inhibited if cells lack Mec1/ATR kinase. Tel1/ATM, in contrast, acts as a suppressor independently of Mec3 and Sgs1. Translocations are also inhibited in cells lacking Dun1 kinase, but not in cells defective in a parallel checkpoint branch defined by Chk1 kinase. While we had previously shown that RAD51 deletion did not inhibit translocation formation, RAD59 deletion led to inhibition comparable to the rad52Δ mutation. A candidate screen of other DNA metabolic factors identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1Δ mutant, becoming even more important for chromosomal stability upon MEC3 deletion. We determined that the C-terminal third of Exo1, harboring mismatch repair protein binding sites and phosphorylation sites, is dispensable for Exo1's roles in chromosomal rearrangement suppression, mutation avoidance and resistance to DNA-damaging agents. Conclusions: Our findings suggest that translocations between related genes can form by Rad59-dependent, Rad51-independent homologous recombination, which is independently suppressed by Sgs1, Tel1, Mec3 and Exo1 but promoted by Dun1 and the telomerase-inhibitor Mec1. We propose a model for the functional interaction between mitotic recombination and the DNA-damage checkpoint in the suppression of chromosomal rearrangements in sgs1Δ cells. http://www.genomeintegrity.com/content/2/1/8 Lillian Doerfler Lorena Harris Emilie Viebranz Kristina Schmidt Genome Integrity 2011, null:8 2011-10-31T00:00:00Z doi:10.1186/2041-9414-2-8 /content/figures/2041-9414-2-8-toc.gif Genome Integrity 2041-9414 ${item.volume} 8 2011-10-31T00:00:00Z XML Cellular senescence is a normal biological process that is initiated in response to a range of intrinsic and extrinsic factors that functions to remove irreparable damage and therefore potentially harmful cells, from the proliferative pool. Senescence can therefore be thought of in beneficial terms as a tumour suppressor. In contrast to this, there is a growing body of evidence suggesting that senescence is also associated with the disruption of the tissue microenvironment and development of a pro-oncogenic environment, principally via the secretion of senescence-associated pro-inflammatory factors. The fraction of cells in a senescent state is known to increase with cellular age and from exposure to various stressors including ionising radiation therefore, the implications of the detrimental effects of the senescent phenotype are important to understand within the context of the increasing human exposure to ionising radiation. This review will discuss what is currently understood about senescence, highlighting possible associations between senescence and cancer and, how exposure to ionising radiation may modify this. http://www.genomeintegrity.com/content/2/1/7 Rebecca Sabin Rhona Anderson Genome Integrity 2011, null:7 2011-08-11T00:00:00Z doi:10.1186/2041-9414-2-7 /content/figures/2041-9414-2-7-toc.gif Genome Integrity 2041-9414 ${item.volume} 7 2011-08-11T00:00:00Z XML Background: Transgenic cattle carrying multiple genomic modifications have been produced by serial rounds of somatic cell chromatin transfer (cloning) of sequentially genetically targeted somatic cells. However, cloning efficiency tends to decline with the increase of rounds of cloning. It is possible that multiple rounds of cloning compromise the genome integrity or/and introduce epigenetic errors in the resulting cell lines, rendering a decline in cloning. To test these possibilities, we performed 9 high density array Comparative Genomic Hybridization (CGH) experiments to test the genome integrity in 3 independent bovine transgenic cell lineages generated from genetic modification and cloning. Our plan included the control hybridizations (self to self) of the 3 founder cell lines and 6 comparative hybridizations between these founders and their derived cell lines with either high or low cloning efficiencies. Results: We detected similar amounts of differences between the control hybridizations (8, 13 and 39 differences) and the comparative analyses of both "high" and "low" cell lines (ranging from 7 to 57 with a mean of ~20). Almost 75% of the large differences (>10 kb) and about 45% of all differences shared the same type (loss or gain) and were located in nearby genomic regions across hybridizations. Therefore, it is likely that they were not true differences but caused by systematic factors associated with local genomic features (e.g. GC contents). Conclusions: Our findings reveal that large copy number variations are less likely to arise during genetic targeting and serial rounds of cloning, fortifying the notion that epigenetic errors introduced from serial cloning may be responsible for the cloning efficiency decline. http://www.genomeintegrity.com/content/2/1/6 George Liu Yali Hou James Robl Yoshimi Kuroiwa Zhongde Wang Genome Integrity 2011, null:6 2011-05-23T00:00:00Z doi:10.1186/2041-9414-2-6 /content/figures/2041-9414-2-6-toc.gif Genome Integrity 2041-9414 ${item.volume} 6 2011-05-23T00:00:00Z XML Background: In mammalian cells gene amplification is a common manifestation of genome instability promoted by DNA double-strand breaks (DSBs). The repair of DSBs mainly occurs through two mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). We previously showed that defects in the repair of DSBs via NHEJ could increase the frequency of gene amplification. In this paper we explored whether a single or a combined defect in DSBs repair pathways can affect gene amplification. Results: We constructed human cell lines in which the expression of RAD54 and/or DNA-PKcs was constitutively knocked-down by RNA interference. We analyzed their radiosensitivity and their capacity to generate amplified DNA. Our results showed that both RAD54 and DNA-PKcs deficient cells are hypersensitive to γ-irradiation and generate methotrexate resistant colonies at a higher frequency compared to the proficient cell lines. In addition, the analysis of the cytogenetic organization of the amplicons revealed that isochromosome formation is a prevalent mechanism responsible for copy number increase in RAD54 defective cells. Conclusions: Defects in the DSBs repair mechanisms can influence the organization of amplified DNA. The high frequency of isochromosome formation in cells deficient for RAD54 suggests that homologous recombination proteins might play a role in preventing rearrangements at the centromeres. http://www.genomeintegrity.com/content/2/1/5 Aurora Ruiz-Herrera Alexandra Smirnova Lela Khoriauli Solomon Nergadze Chiara Mondello Elena Giulotto Genome Integrity 2011, null:5 2011-03-18T00:00:00Z doi:10.1186/2041-9414-2-5 /content/figures/2041-9414-2-5-toc.gif Genome Integrity 2041-9414 ${item.volume} 5 2011-03-18T00:00:00Z XML Background: Fanconi anemia (FA) is a rare autosomal recessive syndrome characterized by developmental abnormalities, progressive bone marrow failure, and predisposition to cancer. The key FA protein FANCD2 crosstalks with members of DNA damage and repair pathways that also play a role at telomeres. Therefore, we investigated whether FANCD2 has a similar involvement at telomeres. Results: We reveal that FANCD2 may perform a novel function separate to the FANCD2/BRCA pathway. This function includes FANCD2 interaction with one of the telomere components, the PARP family member tankyrase-1. Moreover, FANCD2 inhibits tankyrase-1 activity in vitro. In turn, FANCD2 deficiency increases the polyADP-ribosylation of telomere binding factor TRF1. Conclusions: FANCD2 binding and inhibiting tankyrase-1PARsylation at telomeres may provide an additional step within the FA pathway for the regulation of genomic integrity. http://www.genomeintegrity.com/content/2/1/4 Alex Lyakhovich Maria Jose Ramirez Andres Castellanos Maria Castella Amanda Simons Jeffrey Parvin Jordi Surralles Genome Integrity 2011, null:4 2011-02-12T00:00:00Z doi:10.1186/2041-9414-2-4 /content/figures/2041-9414-2-4-toc.gif Genome Integrity 2041-9414 ${item.volume} 4 2011-02-12T00:00:00Z XML Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds. http://www.genomeintegrity.com/content/2/1/3 Li-Jeen Mah Christian Orlowski Katherine Ververis Raja Vasireddy Assam El-Osta Tom Karagiannis Genome Integrity 2011, null:3 2011-01-25T00:00:00Z doi:10.1186/2041-9414-2-3 /content/figures/2041-9414-2-3-toc.gif Genome Integrity 2041-9414 ${item.volume} 3 2011-01-25T00:00:00Z XML Background: Telomeres are protective cap structures at the ends of the linear eukaryotic chromosomes, which provide stability to the genome by shielding from degradation and chromosome fusions. The cap consists of telomere-specific proteins binding to the respective single- and double-stranded parts of the telomeric sequence. In addition to the nucleation of the chromatin structure the telomere-binding proteins are involved in the regulation of the telomere length. However, the telomeric sequences are highly diverged among yeast species. During the evolution this high rate of divergency presents a challenge for the sequence recognition of the telomere-binding proteins. Results: We found that the Saccharomyces castellii protein Rap1, a negative regulator of telomere length, binds a 12-mer minimal binding site (MBS) within the double-stranded telomeric DNA. The sequence specificity is dependent on the interaction with two 5 nucleotide motifs, having a 6 nucleotide centre-to-centre spacing. The isolated DNA-binding domain binds the same MBS and retains the same motif binding characteristics as the full-length Rap1 protein. However, it shows some deviations in the degree of sequence-specific dependence in some nucleotide positions. Intriguingly, the positions of most importance for the sequence-specific binding of the full-length Rap1 protein coincide with 3 of the 4 nucleotides utilized by the 3' overhang binding protein Cdc13. These nucleotides are very well conserved within the otherwise highly divergent telomeric sequences of yeasts. Conclusions: Rap1 and Cdc13 are two very distinct types of DNA-binding proteins with highly separate functions. They interact with the double-stranded vs. the single-stranded telomeric DNA via significantly different types of DNA-binding domain structures. However, we show that they are dependent on coinciding nucleotide positions for their sequence-specific binding to telomeric sequences. Thus, we conclude that during the molecular evolution they act together to preserve a core sequence of the telomeric DNA. http://www.genomeintegrity.com/content/2/1/2 Jenny Rhodin Edso Cecilia Gustafsson Marita Cohn Genome Integrity 2011, null:2 2011-01-14T00:00:00Z doi:10.1186/2041-9414-2-2 /content/figures/2041-9414-2-2-toc.gif Genome Integrity 2041-9414 ${item.volume} 2 2011-01-14T00:00:00Z XML