Source Of Genomic Instability Research Articles

Abstract A04: Phosphorylation and ubiquitylation on the RPA-ssDNA platform promote homologous recombination

Abstract Replication stress is an important source of genome instability in cancer cells. Impediments to replication fork progression stimulate the accumulation of RPA-coated single-stranded DNA (RPA-ssDNA). RPA-ssDNA then recruits and activates a large number of genome maintenance factors including the master checkpoint kinase ATR to protect genomic integrity. Amongst the factors recruited onto RPA-ssDNA, we have previously shown that the E3 ubiquitin ligase PRP19, in addition to its central role as an RNA splicing factor, plays an integral part in the elicitation of the DDR during replication stress. Specifically, the ubiquitin ligase activity of PRP19 on the RPA-ssDNA platform is critical for the activation of the ATR checkpoint kinase and promotes replication fork stability during stress. How exactly is PRP19 tethered to RPA-ssDNA in response to damage and whether its ubiquitin ligase activity on this platform is important for specific DNA repair pathways remains unexplored. Here, we show that RPA ubiquitylation is potently triggered by genotoxic agents which target replication forks. The ubiquitylation of RPA and its interaction with PRP19 both correlate with RPA32 phosphorylation. In fact, we show that ubiquitylated RPA is also phosphorylated. Interestingly, a non-phosphorylatable RPA32 mutant interacts poorly with PRP19 and is not efficiently ubiquitylated. Furthermore, we identify a positively charged pocket on the PRP19 WD40 domain which interacts with RPA during replication stress suggesting that PRP19 may recognize phosphorylated RPA through this surface. Finally, we show that the ubiquitin ligase activity of PRP19 and its ability to interact with RPA through its electropositive pocket are both required for optimal homologous recombination. Our work sheds new light on the regulation of RPA-ssDNA ubiquitylation which is critical for genome stability. We show that a splicing factor, PRP19, reallocates its activity in response to damage through the combined activities of PI3K-like kinases on RPA-ssDNA, highlighting the intricate crosstalk between RNA maturation factors and genome maintenance. We propose a model whereby RPA phosphorylation promotes its ubiquitylation by PRP19 and potentially other ubiquitin ligases and favors error-free repair of DNA breaks via homologous recombination. Because of their roles in replication stress tolerance, targeting mediators of RPA ubiquitylation may synergize with chemotherapeutic agents that inhibit DNA replication thereby improving the efficiency of cancer treatments. Citation Format: Jean-Christophe Dubois, Geneviève Clément, Laurent Cappadocia, Luc Gaudreau, Lee Zou, Alexandre Maréchal. Phosphorylation and ubiquitylation on the RPA-ssDNA platform promote homologous recombination [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A04.

Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats.

Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.

DNA Oncogenic Virus-Induced Oxidative Stress, Genomic Damage, and Aberrant Epigenetic Alterations.

Approximately 20% of human cancers is attributable to DNA oncogenic viruses such as human papillomavirus (HPV), hepatitis B virus (HBV), and Epstein-Barr virus (EBV). Unrepaired DNA damage is the most common and overlapping feature of these DNA oncogenic viruses and a source of genomic instability and tumour development. Sustained DNA damage results from unceasing production of reactive oxygen species and activation of inflammasome cascades that trigger genomic changes and increased propensity of epigenetic alterations. Accumulation of epigenetic alterations may interfere with genome-wide cellular signalling machineries and promote malignant transformation leading to cancer development. Untangling and understanding the underlying mechanisms that promote these detrimental effects remain the major objectives for ongoing research and hope for effective virus-induced cancer therapy. Here, we review current literature with an emphasis on how DNA damage influences HPV, HVB, and EBV replication and epigenetic alterations that are associated with carcinogenesis.

Noncoding Centromeric RNA Expression Impairs Chromosome Stability in Human and Murine Stem Cells.

We analyzed the effect of transcribed noncoding RNA centromeric satellites on chromosome segregation in normal human and murine stem and fibrosarcoma cells. The overexpression of different centromeric alphoid DNAs in all cell lines induced a marked increase in chromosome mis-segregation in anaphase. Overexpression of centromeric mouse minor satellite also increased chromosome instability in the murine stem but not in human cells. Analysis of chromosome segregation in vivo showed disturbances in the mitotic progression, which was frequently unresolved. Live cell imaging revealed that overexpression of centromeric satellites resulted in several different chromosomal morphological errors in the cell nuclei. Our findings correlated with other reports that several centromeric noncoding RNAs are detected in different carcinoma cells and their expression resulted in segregation errors. Our study furnishes further insights into a novel source of genomic instability in human and murine cells. It has recently been shown that noncoding centromeric RNAs are present in some form of cancer, and thus, overexpression of several types of centromeric noncoding RNAs may be useful as a specific maker for neoplastic cells.

Intrastrand triplex DNA repeats in bacteria: a source of genomic instability.

Repetitive nucleic acid sequences are often prone to form secondary structures distinct from B-DNA. Prominent examples of such structures are DNA triplexes. We observed that certain intrastrand triplex motifs are highly conserved and abundant in prokaryotic genomes. A systematic search of 5246 different prokaryotic plasmids and genomes for intrastrand triplex motifs was conducted and the results summarized in the ITxF database available online at http://bioinformatics.uni-konstanz.de/utils/ITxF/. Next we investigated biophysical and biochemical properties of a particular G/C-rich triplex motif (TM) that occurs in many copies in more than 260 bacterial genomes by CD and nuclear magnetic resonance spectroscopy as well as in vivo footprinting techniques. A characterization of putative properties and functions of these unusually frequent nucleic acid motifs demonstrated that the occurrence of the TM is associated with a high degree of genomic instability. TM-containing genomic loci are significantly more rearranged among closely related Escherichia coli strains compared to control sites. In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse. In conclusion we link intrastrand triplex motifs with the induction of genomic instability. We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon.

Abstract 3012: The Snm1B/Apollo DNA nuclease functions in resolution of replication stress and maintenance of genome stability

Abstract A critical aspect of normal cellular processes is the maintenance of genomic stability. Defects in cellular responses to DNA damage can lead to an accumulation of unrepaired or misrepaired lesions and ultimately, increased genome instability. Genomic DNA is constantly damaged through exposure to exogenous agents as well as during endogenous processes, including DNA replication. Progression of the replication fork can be impaired by structural or physical blocks leading to fork stalling. If stalled forks are not properly restarted or repaired, they can collapse, leading to chromosomal damage including breaks, deletions, and translocations. Therefore, replication stress associated DNA damage has been hypothesized as one important source of genome instability associated with cancer initiation and progression. Cells have evolved intricate mechanisms to detect, signal, and resolve stalled replication forks; however, the molecular events involved in these fundamental processes have not been fully defined. My studies are focused on the DNA nuclease SNM1B/Apollo which plays critical roles in repairing stalled or blocked replication forks and also in telomere maintenance. We previously reported that SNM1B functions within the Fanconi anemia (FA) pathway, which is required for efficient stabilization of stalled replication forks. Mutations in the 16 known FA genes cause Fanconi anemia, a human genome instability disorder characterized by cancer predisposition, progressive bone marrow failure, and developmental defects. It is emerging that the SNM1B locus is genomically altered or mutated in a variety of cancer types and has been mapped at a chromosomal breakpoint associated with Wilms tumor. Thus, SNM1B likely plays important roles in the prevention of potential oncogenic chromosomal anomalies; however, its precise functions during DNA repair processes are not well understood. My current research is focused on elucidating the importance of the nuclease activity of SNM1B in stabilizing and repairing stalled replication forks and how these functions are regulated. Interestingly, SNM1B possesses the monoubiquitination consensus sequence, RKQL, found in the key FA protein, FANCD2. I found that substituting arginine for lysine within the consensus sequence results in defective localization of the mutant SNM1B protein to stalled forks as well as impaired FANCD2 recruitment to sites of damage, indicating that this residue has functional importance in the repair of stalled forks. Using a siRNA knockdown approach in human cell lines, I determined that SNM1B depletion significantly increases fork stalling and single-stranded DNA regions, even in unperturbed cells. Altogether, my recent studies led us to propose that functions of the SNM1B nuclease during the repair of stalled replication forks ensures successful replication of the genome and thus, prevents potentially oncogenic chromosomal aberrations. Citation Format: Ishita Das, Jennifer Mason, JoAnn Sekiguchi. The Snm1B/Apollo DNA nuclease functions in resolution of replication stress and maintenance of genome stability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3012. doi:10.1158/1538-7445.AM2015-3012

Zygotic Genome Activation Triggers Chromosome Damage and Checkpoint Signaling in C. elegans Primordial Germ Cells

Recent findings have identified highly transcribed genes as a source of genome instability; however, the degree to which large-scale shifts in transcriptional activity cause DNA damage was not known. One example of a large-scale shift in transcriptional activity occurs during development, when maternal regulators are destroyed and zygotic genome activation (ZGA) occurs. Here, we show that ZGA triggers widespread chromosome damage in the primordial germ cells of the nematode C. elegans. We show that ZGA-induced DNA damage activates a checkpoint response, the damage is repaired by factors required for inter-sister homologous recombination, and topoisomerase II plays a role in generating the damage. These findings identify ZGA as a source of intrinsic genome instability in the germline and suggest that genome destabilization may be a general consequence of extreme shifts in cellular transcriptional load.

Replication stress and cancer.

Genome instability is a hallmark of cancer, and DNA replication is the most vulnerable cellular process that can lead to it. Any condition leading to high levels of DNA damage will result in replication stress, which is a source of genome instability and a feature of pre-cancerous and cancerous cells. Therefore, understanding the molecular basis of replication stress is crucial to the understanding of tumorigenesis. Although a negative aspect of replication stress is its prominent role in tumorigenesis, a positive aspect is that it provides a potential target for cancer therapy. In this Review, we discuss the link between persistent replication stress and tumorigenesis, with the goal of shedding light on the mechanisms underlying the initiation of an oncogenic process, which should open up new possibilities for cancer diagnostics and treatment.

Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control.

DNA replication stress is a source of genomic instability. Here we identify changed mutation rate 1 (Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that Cmr1—together with Mrc1/Claspin, Pph3, the chaperonin containing TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to Cmr1, its human orthologue WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that Cmr1/WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins.

Changes of Osvaldo expression patterns in germline of male hybrids between the species Drosophila buzzatii and Drosophila koepferae.

Hybridization between different genomes is a source of genomic instability, sometimes associated with transposable element (TE) mobilization. Previous work showed that hybridization between the species Drosophila buzzatii and Drosophila koepferae induced mobilization of different (TEs), the Osvaldo retrotransposon being the most unstable. However, we ignore the mechanisms involved in this transposition release in interspecific hybrids. In order to disentangle the mechanisms involved in this process, we performed Osvaldo expression studies in somatic and germinal tissues from hybrids and parental species. There was a trend towards increased Osvaldo expression in the somatic tissues of hybrid females and males, which was always significant in males compared to the parental species D. buzzatii but, not in females compared to maternal species D. koepferae. There were massive changes of Osvaldo expression in the testes, which varied depending on the hybrid generation and family. Moreover, Osvaldo hybridization signals, restricted to the apical and primary spermatocyte regions in parents, occupied broader region in the hybrids. In ovaries, there were no significant differences in Osvaldo expression rates between hybrids and the maternal species D. koepferae. The transcript location was restricted to ovarian nurse cells in both parents and hybrids, undetectable in some hybrids. This research highlights first, the existence of putative complex deregulation mechanisms different between sexes and cell types and second, disruption of Osvaldo activity particularly evident in testes from sterile hybrid males. Deeper studies of the total transcriptome in hybrids and parental species are necessary to gain a better knowledge of the TE deregulation pathways in hybrids.

A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase

Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ(-/-) cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.

Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells.

SUMOylation is a form of post-translational modification involving covalent attachment of SUMO (Small Ubiquitin-like Modifier) polypeptides to specific lysine residues in the target protein. In human cells, there are four SUMO proteins, SUMO1-4, with SUMO2 and SUMO3 forming a closely related subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock. Substantial evidence from studies in yeast has shown that SUMOylation plays an important role in the regulation of DNA replication and repair. Here, we report a proteomic analysis of proteins modified by SUMO2 in response to DNA replication stress in S phase in human cells. We have identified a panel of 22 SUMO2 targets with increased SUMOylation during DNA replication stress, many of which play key functions within the DNA replication machinery and/or in the cellular response to DNA damage. Interestingly, POLD3 was found modified most significantly in response to a low dose aphidicolin treatment protocol that promotes common fragile site (CFS) breakage. POLD3 is the human ortholog of POL32 in budding yeast, and has been shown to act during break-induced recombinational repair. We have also shown that deficiency of POLD3 leads to an increase in RPA-bound ssDNA when cells are under replication stress, suggesting that POLD3 plays a role in the cellular response to DNA replication stress. Considering that DNA replication stress is a source of genome instability, and that excessive replication stress is a hallmark of pre-neoplastic and tumor cells, our characterization of SUMO2 targets during a perturbed S-phase should provide a valuable resource for future functional studies in the fields of DNA metabolism and cancer biology.

Advancement in the Structural Polymorphism of G-Quadruplexes

Genomes contain a large number of putative guanine-rich sequences, specifically on promoter regions, untranslated regions (UTR’s) and telomeres etc. that could form guanine-quadruplexes, and may serve as important structural and regulatory elements. They can also be the source of genomic instability which may lead to cancer, aging and human genetic diseases. Four guanines in the same plane, joined together with Hoogsteen hydrogen bonding, and stacked over one another resulting in guanine tetrads, give rise to an incredible class of G-quadruplexes. An extensive range of G-quadruplex structures is well documented, where they differ in number of strands (uni, bi, or tetramolecular), conformations (parallel, antiparallel or mixed), shapes (chair or basket form), or types of loops (edgewise/ lateral, diagonal, double chain reversal/ propeller, or V-shaped loops) etc. With advancements in the techniques, various new multistranded G-rich DNA structures are enriching the DNA structural databases. The most recent ones are (3+1) G-quadruplex, Tri-G-quadruplex, G-triplex DNA etc. which actually add to the diversity of G-quadruplex structures. Exploring their polymorphism with various biophysical and biochemical techniques has hence become extremely important. This review mainly focuses on the discussion of these unusual and comparatively new polymorphic G-quadruplex DNA structures. The robustness of these unique (3+1) G-quadruplex or G-triplex or tri-G-quadruplex structures actually can be exploited for providing a strong foundation for the designing of structure-specific drugs. Recently, G-quadruplex structures have been quantitatively visualized in human cells by engineering structure-specific antibody. Considering these developments, the mapping of G-quadruplex structures in genome may now be possible, with a goal of controlling the gene functions or other cellular processes, which might be involved in diseases like cancer.

Genomic instability may originate from imatinib-refractory chronic myeloid leukemia stem cells

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.

Loss of Telomere Protection: Consequences and Opportunities

Telomeres are repetitive sequences at the natural ends of linear eukaryotic chromosomes that protect these from recognition as chromosome breaks. Their ability to do so critically depends on the binding of sufficient quantities of functional shelterin, a six-unit protein complex with specific and crucial roles in telomere maintenance and function. Insufficient telomere length, leading to insufficient concentration of shelterin at chromosome ends, or otherwise crippled shelterin function, causes telomere deprotection. While contributing to aging-related pathologies, loss of telomere protection can act as a barrier to tumorigenesis, as dysfunctional telomeres activate DNA-damage-like checkpoint responses that halt cell proliferation or trigger cell death. In addition, dysfunctional telomeres affect cancer development and progression by being a source of genomic instability. Reviewed here are the different approaches that are being undertaken to investigate the mammalian cellular response to telomere dysfunction and its consequences for cancer. Furthermore, it is discussed how current and future knowledge about the mechanisms underlying telomere damage responses might be applied for diagnostic purposes or therapeutic intervention.

Genomic Instability in CML-CP originates From the Most Primitive Imatinib-Refractory Leukemia Stem Cells

AbstractAbstract 909Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in the appearance of clones carrying BCR-ABL1 kinase mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or those harboring additional chromosomal aberrations, eventually leading to disease relapse and/or malignant progression to blast phase (CML-BP) [Skorski, T., Leukemia and Lymphoma, 2011]. We found that Lin−CD34+CD38− human leukemia stem cells (huLSCs), including the quiescent sub-population, and Lin−CD34+CD38+ human leukemia progenitor cells (huLPCs) accumulate high levels of reactive oxygen species (ROS) resulting in numerous oxidative DNA lesions such as 8-oxoguanine (8-oxoG) and DNA double-strand breaks (DSBs) [Nieborowska-Skorska, Blood, 2012]. huLSCs and huLPCs treated with TKIs continue to exhibit ROS-induced oxidative DNA damage suggesting the persistence of genomic instability in TKI-treated patients. Furthermore, genomic instability in TKI-refractory huLSCs and TKI-sensitive huLPCs may have a varying impact on disease progression and determining novel treatment modalities. To determine if TKI-refractory huLSCs are a source of genomic instability we employed a tetracycline-inducible murine model of CML-CP: SCLtTA/p210BCR-ABL1. Mice exhibiting CML-CP -like disease demonstrated splenomegaly, leukocytosis, and expansion of mature Gr1+/CD11b+ cells. ROS were elevated in Lin−c-Kit+Sca-1+ cells (muLSCs), but not Lin−c-Kit+Sca-1− cells (muLPCs), which was associated with higher mRNA expression of BCR-ABL1 in muLSCs. In addition, ROS levels were directly proportional to BCR-ABL1 kinase expression in transduced CD34+ human hematopoietic cells, thus confirming the “dosage-dependent” effect of BCR-ABL1 on ROS. Among the Lin−c-Kit+Sca-1+ cells, enhanced ROS were detected in TKI-refractory quiescent muLSCs, in CD34−Flt3− long-term and CD34+Flt3− short-term muLSCs, and also in CD34+Flt3+ multipotent progenitors. High levels of ROS in muLSCs were accompanied by aberrant expression of genes regulating ROS metabolism (mitochondrial electron transport, oxidative phosphorylation, hydrogen peroxide synthesis, and detoxification). In addition, muLSCs, including the quiescent sub-population, displayed high levels of oxidative DNA lesions (8-oxoG, and DSBs). ROS-induced oxidative DNA damage in muLSCs was accompanied by genomic instability in CML-CP –like mice, which accumulated a broad range of genetic aberrations recapitulating the heterogeneity of sporadic mutations detected in TKI-naive CML-CP patients. These aberrations include TKI-resistant BCR-ABL1 kinase mutations, deletions in Ikzf1 and Trp53 and additions in Zfp423 and Idh1 genes, which have been associated with CML-CP relapse and progression to CML-BP. Imatinib caused only modest inhibition of ROS and oxidative DNA damage in TKI-refractory muLSCs. In concordance, CML-CP –like mice treated with imatinib continued to accumulate genomic aberrations. Since BCR-ABL1(K1172R) kinase-dead mutant expressed in CD34+ human hematopoietic cells did not enhance ROS, it suggests that BCR-ABL1 kinase-independent mechanisms contribute to genomic instability. In summary, we postulate that ROS-induced oxidative DNA damage resulting in genetic instability may originate in the most primitive TKI-refractory huLSCs in TKI-naive and TKI-treated patients. Disclosures:Lange:Novartis: Honoraria, Research Funding. Müller:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Koschmieder:Novartis / Novartis Foundation: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Noncanonical Mismatch Repair as a Source of Genomic Instability in Human Cells

Mismatch repair (MMR) is a key antimutagenic process that increases the fidelity of DNA replication and recombination. Yet genetic experiments showed that MMR is required for antibody maturation, a process during which the immunoglobulin loci of antigen-stimulated B cells undergo extensive mutagenesis and rearrangements. In an attempt to elucidate the mechanism underlying the latter events, we set out to search for conditions that compromise MMR fidelity. Here, we describe noncanonical MMR (ncMMR), a process in which the MMR pathway is activated by various DNA lesions rather than by mispairs. ncMMR is largely independent of DNA replication, lacks strand directionality, triggers PCNA monoubiquitylation, and promotes recruitment of the error-prone polymerase-η to chromatin. Importantly, ncMMR is not limited to B cells but occurs also in other cell types. Moreover, it contributes to mutagenesis induced by alkylating agents. Activation of ncMMR may therefore play a role in genomic instability and cancer.

Transcription as a source of genome instability

Alterations in genome sequence and structure contribute to somatic disease, affect the fitness of subsequent generations and drive evolutionary processes. The crucial roles of highly accurate replication and efficient repair in maintaining overall genome integrity are well-known, but the more localized stability costs that are associated with transcribing DNA into RNA molecules are less appreciated. Here we review the diverse ways in which the essential process of transcription alters the underlying DNA template and thereby modifies the genetic landscape.

DNA G‐quadruplex: structure, function and human disease

The role of non-B DNA in the function and stability of genomes has become generally appreciated in recent years. It is now evident that DNA also encodes for spatial structures that are involved in gene regulation, replication and recombination. There are several types of secondary structures in non-B DNA. Among these noncanonical structures are hairpins and cruciforms, intramolecular triplexes or H-DNA, left-handed Z-DNA and guanine-rich repeats which have the capacity to adopt G-quadruplex (G4-DNA); the latter structure is also referred to in the literature as G-tetraplex. G4-DNA consists of a hydrogen-bonded self-assembly of four guanine bases, paired by Hoogsteen bonding, which forms planar arrangements, the G-quartets. Charge coordination by monovalent cations stabilizes G-quartet stacking, resulting in intramolecular or intermolecular association of four DNA strands in a parallel or antiparallel orientation. Genomes contain a high number of G-rich sequences that could form G4-DNA and this structure may serve important regulatory and structural functions; in addition, it can be the source of genomic instability which may lead to cancer, aging and human genetic diseases. In silico analysis using different computational methods, as well as laboratory experiments, indicates that many G-rich regions of chromosomes – rDNA, single-copy genes, recombination sites like those involved in immunoglobulin class-switching and repetitive sequences including satellite and telomeric DNA sequences – have the potential to form G4-DNA structures. Interestingly, RNA is also capable of forming G4 structures even more stable than those of DNA. In this sense, it is tempting to speculate that RNA G-quadruplexes could play a role in translation regulation, in a manner analogous to G4-DNA and transcription. In the human genome, the number of sites with the potential to form G-quadruplex is surprisingly high. Some of these sites are within genes and are potential threats to genome stability because they can alter the DNA architecture and interfere with normal DNA processes, such as replication and transcription. Although G4-DNA has been rigorously studied in vitro, whether this structure actually forms in vivo and what its cellular roles might be, remains unclear. However, recent advances have made a persuasive case for the existence of G4-DNA in living cells and its participation in the regulation of processes as varied as telomere maintenance, transcription, recombination and ribosome biogenesis. This minireview series deals with the structural and functional characteristics of G4-DNA, as well as its implication in human disease. In the first minireview, Huppert gives an introduction to the structure and genomic distribution of putative G4-DNA-forming sequences and creates a theoretical background for the other two articles in the series. He discusses the computational approaches used to predict them on a genomic scale, and how the information derived can be combined with experiments to understand their biological functions. In the second minireview, Brooks, Kendrick & Hurley examine the diversity of G4 structures and i-motifs in promoter elements and attempt to categorize the different types of arrangements in which they are found. The third minireview, by Wu & Brosh, provocatively summarizes the presumed in vivo role of G4-nucleic acid structures and discusses the consequences of human genetic defects affecting the different enzymes that manipulate G-quadruplex structures. They present all the circumstantial evidence suggesting that G-quadruplex nucleic acid structures, which have been demonstrated unambiguously to be formed on specific sequences in naked DNA in vitro, do exist in vivo in the chromatin-embedded genomic DNA. Finally, they discuss the connections of G-quadruplexes to human genetic diseases and cancer.

Abstract B106: Nedd9 null status initially delays mammary tumor growth, then selects for hyper-aggressive mammary tumors

Abstract Altered expression of the HEF1/CAS-L/NEDD9 scaffolding protein has recently emerged as a candidate mechanism controlling tumor development, progression and metastasis. While some studies have identified elevated NEDD9 expression as pro-metastatic, other works have suggested a negative role in tumor progression. We have recently reported that the Nedd9 null genetic background significantly limits mammary tumor development in the MMTV-polyoma virus middle T (PyVmT) genetic model. Late-developing tumors are characterized by depressed activation of proteins including AKT, SRC, FAK, and ERK, emphasizing an important role of NEDD9 as a scaffolding protein for these pro-oncogenic proteins. However, NEDD9 is also regulates activation of the Aurora-A mitotic kinase (AurA). In vitro, knockdown of NEDD9 reduces AurA activation, suggesting in vivo loss of NEDD9 might be a potential source of genomic instability. We have evaluated a panel of cell lines derived from mammary tumors of MMTV-PyVmT;Nedd9−/− mice. Nedd9−/− cells injected orthotopically in mammary fat pads of SCID mice exhibited significantly faster growth of mammary tumors, and in a tail vein injection assay yielded a dramatically enhanced metastasis to the lungs. As hypothesized, Nedd9−/− cell lines had lower AurA kinase activity than Nedd9+/+ cells, and also had evidence of centrosomal and spindle abnormalities associated with deficient AurA function. Interestingly, the sensitivity of Nedd9−/− cells to small molecule inhibitors targeted at specific partner kinases and to chemotherapeutic agents used in breast cancer notably differed from that of Nedd9+/+ cells. Together, these data support the use of NEDD9 expression as a novel biomarker in establishing prognosis and drug responsiveness of tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B106.