Gross Chromosomal Rearrangements Research Articles

The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.

Two sequential cleavage reactions on cruciform DNA structures cause palindrome-mediated chromosomal translocations

Gross chromosomal rearrangements (GCRs), such as translocations, deletions or inversions, are often generated by illegitimate repair between two DNA breakages at regions with nucleotide sequences that might potentially adopt a non-B DNA conformation. We previously established a plasmid-based model system that recapitulates palindrome-mediated recurrent chromosomal translocations in humans, and demonstrated that cruciform DNA conformation is required for the translocation-like rearrangements. Here we show that two sequential reactions that cleave the cruciform structures give rise to the translocation: GEN1-mediated resolution that cleaves diagonally at the four-way junction of the cruciform and Artemis-mediated opening of the subsequently formed hairpin ends. Indeed, translocation products in human sperm reveal the remnants of this two-step mechanism. These two intrinsic pathways that normally fulfil vital functions independently, Holliday-junction resolution in homologous recombination and coding joint formation in rearrangement of antigen-receptor genes, act upon the unusual DNA conformation in concert and lead to a subset of recurrent GCRs in humans.

Recombination-restarted replication makes inverted chromosome fusions at inverted repeats

SummaryImpediments to DNA replication are known to induce gross chromosomal rearrangements (GCR) and copy number variations (CNV). GCRs/CNVs underlie human genomic disorders1 and are a feature of cancer2. During cancer development environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs/CNVs are proposed to contribute to cancer development and therapy resistance3. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination (HR) or microhomology-primed re-initiation (FoSTeS/MMBIR)4,5. Here we ascertain the consequences of replication with a fork restarted by HR. We identify a new mechanism of chromosomal rearrangement: recombination-restarted forks have an exceptionally high propensity to execute a U-turn at small inverted repeats (up to 1:40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer and to non-recurrent CNVs in genomic disorders.

Repair of DNA Double Strand Breaks by RNA/DNA Patches in U937 Cells.

Abstract 2375It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR), including chromosomal amplifications, deletions, inversions, and translocations. To study repair of DNA DSB in vivo, we generated a vector containing the EF1a promoter driving expression of the herpes simplex thymidine kinase (HsTK); interspersed between the EF1a promoter and Hstk cDNA was the recognition site for the rare-cutting meganuclease I-SceI. This vector was electroporated into U937 cells, and a clone containing a single copy, of the EF1aTK vector, designated F5, was isolated. Transfection of the F5 cells with an I-SceI expression vector, followed by ganciclovir selection, identified clones that had lost expression of HsTK. We recovered no GCR with this approach; most of the GCV-resistant clones had partial or complete deletions of the EF1a promoter, the HsTK cDNA, or both. However we noted that ∼5–10% of repair elements involved insertions of DNA sequences derived from distant regions of the genome; the length of the inserted fragment varied from 47 to 756 bp. Surprisingly, all of the inserted fragments were derived from gene and/or retrotransposon repeat elements such as LINE (Long Interspersed Nuclear Element) or SINE (Short Interspersed Nuclear Element) sequences. Therefore, we hypothesized that the inserted sequences used to “patch” the DNA DSB could be based on reverse transcription of an RNA template. Since sequences derived from human RNA and human genomic DNA are identical (with the exception of RNA splice events, poly-A tails, and RNA-edited nucleotides), we co-transfected the F5 cells with murine RNA and an I-SceI expression vector to test the hypothesis that the patches at the DNA DSB sites could be derived from RNA. After hygromycin selection for successfully transfected cells, genomic DNA was isolated, and amplified with primers that flanked the I-SceI cleavage site. DNA fragments of 500–1000bp (larger than the size of the uncleaved EF!aTK PCR product, which was 400 bp) containing insertions at the I-SceI site were isolated, subcloned, and sequenced. We identified 51 independent sequences which had insertions of 23–266 bp at I-SceI cleavage site. Of these 51 insertions, 4 were vector capture events (derived form the expression vector), and 4 were too short to identify unambiguously. Of the remaining 43 insertions, 39 were derived from a single genomic loci, and 4 samples contained identifiable sequences from 2 distinct genomic regions. The sequences were derived from 16 of the 24 human chromosomes, with no clear preference for any specific chromosome. 62% of the sequences were found to contain sequences from a transcribed gene region, and 64% contained repeat sequences such as LINE, SINE, or LTR. The involvement of retrotransposon sequences, which are known to be reverse transcribed and integrated into the genome, supports the hypothesis that at least some of the insertions may be templated from RNA. Furthermore, 45 of the 47 inserted sequences were derived from endogenous human sequences; however 2 insertions matched to mouse sequences, and were derived from the co-transfected mouse RNA. One sample matched an intronic region of murine Vwa3b, which we confirmed was highly expressed in the mouse cells used to harvest the RNA used for the co-transfection. A second sample was not a gene sequence but contained a LINE element.Taken together, these findings demonstrate that I-SceI-induced DNA DSB can be repaired by “patches” derived from distant regions of the genome. This is the first systematic study focused on captured sequences at DNA DSB sites in the mammalian genome, and suggests that transcribed mRNA and retrotransposons can play an important role in the repair of DNA DSB and preservation of genomic integrity. Finally, the observation that I-SceI-induced DNA DSBs are often repaired by insertions suggests that these insertions could be mistaken for chromosome translocations, if only one “side” of the DNA DSB is sequenced. Disclosures:No relevant conflicts of interest to declare.

Bioinformatic identification of genes suppressing genome instability

Unbiased forward genetic screens for mutations causing increased gross chromosomal rearrangement (GCR) rates in Saccharomyces cerevisiae are hampered by the difficulty in reliably using qualitative GCR assays to detect mutants with small but significantly increased GCR rates. We therefore developed a bioinformatic procedure using genome-wide functional genomics screens to identify and prioritize candidate GCR-suppressing genes on the basis of the shared drug sensitivity suppression and similar genetic interactions as known GCR suppressors. The number of known suppressors was increased from 75 to 110 by testing 87 predicted genes, which identified unanticipated pathways in this process. This analysis explicitly dealt with the lack of concordance among high-throughput datasets to increase the reliability of phenotypic predictions. Additionally, shared phenotypes in one assay were imperfect predictors for shared phenotypes in other assays, indicating that although genome-wide datasets can be useful in aggregate, caution and validation methods are required when deciphering biological functions via surrogate measures, including growth-based genetic interactions.

Stimulation of Gross Chromosomal Rearrangements by the Human CEB1 and CEB25 Minisatellites in Saccharomyces cerevisiae Depends on G-Quadruplexes or Cdc13

Genomes contain tandem repeats that are at risk of internal rearrangements and a threat to genome integrity. Here, we investigated the behavior of the human subtelomeric minisatellites HRAS1, CEB1, and CEB25 in Saccharomyces cerevisiae. In mitotically growing wild-type cells, these GC–rich tandem arrays stimulate the rate of gross chromosomal rearrangements (GCR) by 20, 1,620, and 276,000-fold, respectively. In the absence of the Pif1 helicase, known to inhibit GCR by telomere addition and to unwind G-quadruplexes, the GCR rate is further increased in the presence of CEB1, by 385-fold compared to the pif1Δ control strain. The behavior of CEB1 is strongly dependent on its capacity to form G-quadruplexes, since the treatment of WT cells with the Phen-DC3 G-quadruplex ligand has a 52-fold stimulating effect while the mutation of the G-quadruplex-forming motif reduced the GCR rate 30-fold in WT and 100-fold in pif1Δ cells. The GCR events are telomere additions within CEB1. Differently, the extreme stimulation of CEB25 GCR depends on its affinity for Cdc13, which binds the TG-rich ssDNA telomere overhang. This property confers a biased orientation-dependent behavior to CEB25, while CEB1 and HRAS1 increase GCR similarly in either orientation. Furthermore, we analyzed the minisatellites‚ distribution in the human genome and discuss their potential role to trigger subtelomeric rearrangements.

Gross chromosomal rearrangement mediated by DNA replication in stressed cells: evidence from Escherichia coli

Gross chromosomal rearrangements (GCRs), or changes in chromosome structure, play central roles in evolution and are central to cancer formation and progression. GCRs underlie copy number variation (CNV), and therefore genomic disorders that stem from CNV. We study amplification in Escherichia coli as a model system to understand mechanisms and circumstances of GCR formation. Here, we summarize observations that led us to postulate that GCR occurs by a replicative mechanism as part of activated stress responses. We report that we do not find RecA to be downregulated by stress on a population basis and that constitutive expression of RecA does not inhibit amplification, as would be expected if downregulation of RecA made cells permissive for nonhomologous recombination. Strains deleted for the genes for three proteins that inhibit RecA activity, psiB, dinI, and recX, all show unaltered amplification, suggesting that if they do downregulate RecA indirectly, this activity does not promote amplification.

Rapid Analysis of Saccharomyces cerevisiae Genome Rearrangements by Multiplex Ligation–Dependent Probe Amplification

Aneuploidy and gross chromosomal rearrangements (GCRs) can lead to genetic diseases and the development of cancer. We previously demonstrated that introduction of the repetitive retrotransposon Ty912 onto a nonessential chromosome arm of Saccharomyces cerevisiae led to increased genome instability predominantly due to increased rates of formation of monocentric nonreciprocal translocations. In this study, we adapted Multiplex Ligation–dependent Probe Amplification (MLPA) to analyze a large numbers of these GCRs. Using MLPA, we found that the distribution of translocations induced by the presence of Ty912 in a wild-type strain was nonrandom and that the majority of these translocations were mediated by only six translocation targets on four different chromosomes, even though there were 254 potential Ty-related translocation targets in the S. cerevisiae genome. While the majority of Ty912-mediated translocations resulted from RAD52-dependent recombination, we observed a number of nonreciprocal translocations mediated by RAD52-independent recombination between Ty1 elements. The formation of these RAD52-independent translocations did not require the Rad51 or Rad59 homologous pairing proteins or the Rad1–Rad10 endonuclease complex that processes branched DNAs during recombination. Finally, we found that defects in ASF1-RTT109–dependent acetylation of histone H3 lysine residue 56 (H3K56) resulted in increased accumulation of both GCRs and whole-chromosome duplications, and resulted in aneuploidy that tended to occur simultaneously with GCRs. Overall, we found that MLPA is a versatile technique for the rapid analysis of GCRs and can facilitate the genetic analysis of the pathways that prevent and promote GCRs and aneuploidy.

PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells

Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1 ΔB hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1 ΔB hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing.

Multiple Regulatory Mechanisms to Inhibit Untimely Initiation of DNA Replication Are Important for Stable Genome Maintenance

Genomic instability is a hallmark of human cancer cells. To prevent genomic instability, chromosomal DNA is faithfully duplicated in every cell division cycle, and eukaryotic cells have complex regulatory mechanisms to achieve this goal. Here, we show that untimely activation of replication origins during the G1 phase is genotoxic and induces genomic instability in the budding yeast Saccharomyces cerevisiae. Our data indicate that cells preserve a low level of the initiation factor Sld2 to prevent untimely initiation during the normal cell cycle in addition to controlling the phosphorylation of Sld2 and Sld3 by cyclin-dependent kinase. Although untimely activation of origin is inhibited on multiple levels, we show that deregulation of a single pathway can cause genomic instability, such as gross chromosome rearrangements (GCRs). Furthermore, simultaneous deregulation of multiple pathways causes an even more severe phenotype. These findings highlight the importance of having multiple inhibitory mechanisms to prevent the untimely initiation of chromosome replication to preserve stable genome maintenance over generations in eukaryotes.

A Genetic and Structural Study of Genome Rearrangements Mediated by High Copy Repeat Ty1 Elements

Ty elements are high copy number, dispersed repeated sequences in the Saccharomyces cerevisiae genome known to mediate gross chromosomal rearrangements (GCRs). Here we found that introduction of Ty912, a previously identified Ty1 element, onto the non-essential terminal region of the left arm of chromosome V led to a 380-fold increase in the rate of accumulating GCRs in a wild-type strain. A survey of 48 different mutations identified those that either increased or decreased the rate of Ty-mediated GCRs and demonstrated that suppression of Ty-mediated GCRs differs from that of both low copy repeat sequence- and single copy sequence-mediated GCRs. The majority of the Ty912-mediated GCRs observed were monocentric nonreciprocal translocations mediated by RAD52-dependent homologous recombination (HR) between Ty912 and a Ty element on another chromosome arm. The remaining Ty912-mediated GCRs appeared to involve Ty912-mediated formation of unstable dicentric translocation chromosomes that were resolved by one or more Ty-mediated breakage-fusion-bridge cycles. Overall, the results demonstrate that the Ty912-mediated GCR assay is an excellent model for understanding mechanisms and pathways that suppress genome rearrangements mediated by high copy number repeat sequences, as well as the mechanisms by which such rearrangements occur.

Ultrafine anaphase bridges, broken DNA and illegitimate recombination induced by a replication fork barrier

Most DNA double-strand breaks (DSBs) in S- and G2-phase cells are repaired accurately by Rad51-dependent sister chromatid recombination. However, a minority give rise to gross chromosome rearrangements (GCRs), which can result in disease/death. What determines whether a DSB is repaired accurately or inaccurately is currently unclear. We provide evidence that suggests that perturbing replication by a non-programmed protein–DNA replication fork barrier results in the persistence of replication intermediates (most likely regions of unreplicated DNA) into mitosis, which results in anaphase bridge formation and ultimately to DNA breakage. However, unlike previously characterised replication-associated DSBs, these breaks are repaired mainly by Rad51-independent processes such as single-strand annealing, and are therefore prone to generate GCRs. These data highlight how a replication-associated DSB can be predisposed to give rise to genome rearrangements in eukaryotes.

The contribution of the S-phase checkpoint genes MEC1 and SGS1 to genome stability maintenance in Candida albicans

Genome rearrangements, a common feature of Candida albicans isolates, are often associated with the acquisition of antifungal drug resistance. In Saccharomyces cerevisiae, perturbations in the S-phase checkpoints result in the same sort of Gross Chromosomal Rearrangements (GCRs) observed in C. albicans. Several proteins are involved in the S. cerevisiae cell cycle checkpoints, including Mec1p, a protein kinase of the PIKK (phosphatidyl inositol 3-kinase-like kinase) family and the central player in the DNA damage checkpoint. Sgs1p, the ortholog of BLM, the Bloom’s syndrome gene, is a RecQ-related DNA helicase; cells from BLM patients are characterized by an increase in genome instability. Yeast strains bearing deletions in MEC1 or SGS1 are viable (in contrast to the inviability seen with loss of MEC1 in S. cerevisiae) but the different deletion mutants have significantly different phenotypes. The mec1Δ/Δ colonies have a wild-type colony morphology, while the sgs1Δ/Δ mutants are slow-growing, producing wrinkled colonies with pseudohyphal-like cells. The mec1Δ/Δ mutants are only sensitive to ethylmethane sulfonate (EMS), methylmethane sulfonate (MMS), and hydroxyurea (HU) but the sgs1Δ/Δ mutants exhibit a high sensitivity to all DNA-damaging agents tested. In an assay for chromosome 1 integrity, the mec1Δ/Δ mutants exhibit an increase in genome instability; no change was observed in the sgs1Δ/Δ mutants. Finally, loss of MEC1 does not affect sensitivity to the antifungal drug fluconazole, while loss of SGS1 leads to an increased susceptibility to fluconazole. Neither deletion elevated the level of antifungal drug resistance acquisition.

Sgs1 Truncations Induce Genome Rearrangements but Suppress Detrimental Effects of BLM Overexpression in Saccharomyces cerevisiae

RecQ-like DNA helicases are conserved from bacteria to humans. They perform functions in the maintenance of genome stability, and their mutation is associated with cancer predisposition and premature aging syndromes in humans. Here, a series of C-terminal deletions and point mutations of Sgs1, the only RecQ-like helicase in yeast, show that the Helicase/RNase D C-terminal domain and the Rad51 interaction domain are dispensable for Sgs1's role in suppressing genome instability, whereas the zinc-binding domain and the helicase domain are required. BLM expression from the native SGS1 promoter had no adverse effects on cell growth and was unable to complement any sgs1Δ defects. BLM overexpression, however, significantly increased the rate of accumulating gross-chromosomal rearrangements in a dosage-dependent manner and greatly exacerbated sensitivity to DNA-damaging agents. Co-expressing sgs1 truncations of up to 900 residues, lacking all known functional domains of Sgs1, suppressed the hydroxyurea sensitivity of BLM-overexpressing cells, suggesting a functional relationship between Sgs1 and BLM. Protein disorder prediction analysis of Sgs1 and BLM was used to produce a functional Sgs1–BLM chimera by replacing the N-terminus of BLM with the disordered N-terminus of Sgs1. The functionality of this chimera suggests that it is the disordered N-terminus, a site of protein binding and posttranslational modification, that confers species specificity to these two RecQ-like proteins.

Determination of Gross Chromosomal Rearrangement Rates

Cells devote a significant amount of metabolism to maintaining the stability of their genome and to preventing inappropriate chromosomal rearrangements that are characteristic of many cancers. A simple genetic assay using haploid derivatives of the yeast Saccharomyces cerevisiae provides a means to quantitatively measure the rate at which gross chromosomal rearrangements (GCRs) accumulate in different genetic backgrounds. This assay measures the rate of simultaneous inactivation of CAN1 and URA3 markers placed on a nonessential end of a yeast chromosome and in principle can be implemented in any haploid strain. Rearrangements detected with this assay include broken chromosomes healed by de novo telomere additions and a spectrum of inter- and intrachromosomal fusion events. The GCR assay allows for detailed analysis of the contributions of individual genes and different pathways in the suppression of genomic instability.

Post-Replication Repair Suppresses Duplication-Mediated Genome Instability

RAD6 is known to suppress duplication-mediated gross chromosomal rearrangements (GCRs) but not single-copy sequence mediated GCRs. Here, we found that the RAD6- and RAD18-dependent post-replication repair (PRR) and the RAD5-, MMS2-, UBC13-dependent error-free PRR branch acted in concert with the replication stress checkpoint to suppress duplication-mediated GCRs formed by homologous recombination (HR). The Rad5 helicase activity, but not its RING finger, was required to prevent duplication-mediated GCRs, although the function of Rad5 remained dependent upon modification of PCNA at Lys164. The SRS2, SGS1, and HCS1 encoded helicases appeared to interact with Rad5, and epistasis analysis suggested that Srs2 and Hcs1 act upstream of Rad5. In contrast, Sgs1 likely functions downstream of Rad5, potentially by resolving DNA structures formed by Rad5. Our analysis is consistent with models in which PRR prevents replication damage from becoming double strand breaks (DSBs) and/or regulates the activity of HR on DSBs.

Elg1 (Enhanced level of genome instability 1) has an essential role in early development and suppressing tumorigenesis

IntroductionHigh levels of chromosome rearrangements known as gross chromosomal rearrangements (GCRs) such as translocations, deletion of chromosome arm, interstitial deletions, inversions and gene amplification, have been reported in many cancers. Multiple pathways, such as S‐phase checkpoints, DNA replication, chromatin remodeling, and telomere maintenance suppress the GCR formation. Elg1 was discovered during the genome wide screening of yeast S. cerevisiae as a suppressor of GCR formation. Elg1 is a suppressor of genomic instability and functions in DNA repair in yeast and human.ObjectivesTo investigate whether the loss of Elg1 in mouse a in vivo model system causes any genomic disorder and null Elg1 MEF cells shows any Genomic instability phenotypes.ResultsAbsence of Elg1 in mice caused early embryonic lethality and zebra fish Elg1 MO embryos also has developmental problem suggesting Elg1 has an essential role during early development. This embryonic developmental defect rescued in absence of p53 in zebrafish and developed normal. The heterozygous mice developed tumors including sarcoma, carcinoma, and adenoma in various organs from 11 month after birth. The Elg1+/mMEFs and Elg1+/− chicken DT 40 cells show high level of DN A damage sensitivity as well as chromosomal instability in respond to DNA damage. High levels of genomic rearrangements were observed in tumors developed in the Elg1+/mmice by comparative genome hybridization. The microarray and pathway analysis of tumors from the Elg1 heterozygous mice showed the upregulation of Smad independent Tgf‐β pathway.ConclusionsTaken together, Elg1 has an essential role in early development that appears to have a close link to suppress genomic instability and ultimately tumorigenesis.

Nuclear Receptor-Induced Chromosomal Proximity and DNA Breaks Underlie Specific Translocations in Cancer

Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded androgen receptor (AR) first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic activities induced by genotoxic stress and liganded AR, including activation-induced cytidine deaminase and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymes synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by nonhomologous end joining pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded nuclear receptor and genotoxic stress underlies nonrandom tumor translocations, which may function in many types of tumors and pathological processes.

Efficient repair of DNA double-strand breaks in malignant cells with structural instability

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I- SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1α) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I- SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1α promoter from the TK gene, or deletion of either the EF1α promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I- SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I- SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.

The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.