Gross Chromosomal Rearrangements Formation Research Articles

Abstract KP01: Identification of genes and pathways that prevent genome instability

Abstract The genome of many cancers is characterized by ongoing genome instability; however, the causes of genome instability in cancer are not well understood. To understand the control genome instability, we developed three types of genetic assays for measuring the rate of accumulating gross chromosomal rearrangements (GCRs) in the yeast Saccharomyces cerevisiae - one that detects GCRs containing breakpoints in single copy sequences, a second that detects GCRs mediated by segmental duplications, and a third that detects GCRs mediated by Ty elements which are similar to mammalian LINE elements. Initial studies characterizing these three types of assays found that there are specific pathways that suppress duplication-mediated GCRs and identified sequences that are hotspots for the formation of GCRs. We have subsequently used these three assays to perform a genetic screen of the non-essential genes in S. cerevisiae to identify the genetic network that acts to prevent genome instability. This screen involved three steps: a novel bioinformatics screen to identify and prioritize candidate genome instability suppressing genes; a biological genetic screen to identify which of the predicted genes actually suppress genome instability; and a biological genetic screen to identify interacting genes. As a result of these screens, we have identified 184 genes in which defects cause increased GCRs and an additional 459 genes in which mutations only cause increased GCRs when combined with a second mutation thus defining multiple pathways that prevent GCRs. Control experiments indicated that this approach led to the identification of greater than 95% of the non-essential genes that act to suppress genome instability. In a second screen, we identified 44 essential genes in which defects caused increased GCRs. We are now beginning to use the S. cerevisiae genome instability suppressing genes to predict the mammalian genome instability genetic suppressing network. Citation Format: Richard Kolodner, PhD. Identification of genes and pathways that prevent genome instability [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr KP01.

Template homology determines the genetics and mechanisms of gross chromosomal rearrangements in S. cerevisiae (736.11)

Double strand breaks (DSBs) can lead to genome instability and cell death, and must be faithfully repaired. In Saccharomyces cerevisiae, DSBs are primarily repaired via homologous recombination (HR), which uses homologous sequence as a template for repair. Improper repair can result in gross chromosomal rearrangements (GCRs) such as translocations. Aberrant HR may occur when a template sequence with imperfect homology is used instead of completely homologous sequence. It is not known how divergent homologous sequences are selected as targets for non‐allelic HR, or which pathways suppress the formation of such GCRs. To examine this, we devised 3 separate genetic assays to detect GCRs mediated by a distinct sequence with imperfect homology to the rest of the genome. The assays differ from each other in both percent homology to their target sequences and number of potential rearrangement targets. We find that the GCR rates for these assays differ from each other as well as from existing assays. Additionally, the GCRs detected by the 3 assays exhibit diverse genetic requirements, responding differently to mutations affecting the HR pathways, HR between divergent sequences, and the DNA replication checkpoint. Finally, we characterized the products of the GCRs observed in the 3 assays. These results suggest that the homology between the recombination substrate and its rearrangement targets influences the mechanisms and genetics of GCR formation and the GCR spectrum.Grant Funding Source: NIH Grant GM26017

Genomic instability and repair mediated by common repeated sequences

If you happen to find a suspicious cell, say, in your soup, you may recognize to what species it belongs simply by looking at its chromosome configuration (karyotype) under the microscope. This approach works because most cells of most organisms have a stable genome, and gross chromosomal rearrangements (GCRs) such as translocations, inversions, and deletions of big chunks of chromosomes are relatively rare. This is in stark contrast to cancer cells, which exhibit abundant GCRs (1). When the borders of such rearrangements are mapped, they often belong to repetitive sequences, which are scattered across the genome of all organisms. Tandem repetitive sequences are also present at the ends of the eukaryotic chromosomes, forming the telomeres, which help replicate the genome and protect it from degradation (2). Interestingly, telomeric repeats can also be found at internal positions along the chromosomes in many organisms. These interstitial telomeric sequences (ITSs) often colocalize with chromosomal fragile sites and with endpoints of GCRs (3, 4). However, little is known about the mechanisms responsible for genome instability at interstitial telomeric sequences. In PNAS, Aksenova et al. (5) present a study in which the power of yeast genetics is harnessed to address this particular question. Using a sophisticated genetic trap, the authors measure the rate of GCR formation and characterize the molecular mechanisms leading to them. The results show a surprisingly high level of recombinational activity involving these repeated sequences.

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.

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.

Specific pathways prevent duplication-mediated genome rearrangements

SUMMARYWe have investigated the ability of different regions of the left arm of Saccharomyces cerevisiae chromosome V to participate in the formation of gross chromosomal rearrangements (GCRs). We found that the 4.2 kb HXT13 DSF1 region sharing divergent homology with chromosomes IV, X, and XIV, similar to mammalian segmental duplications, was “at-risk” for participating in duplication-mediated GCRs generated by homologous recombination. Numerous genes and pathways, including SGS1, TOP3, RMI1, SRS2, RAD6, SLX1, SLX4, SLX5, MSH2, MSH6, RAD10 and the DNA replication stress checkpoint requiring MRC1 and TOF1 were highly specific for suppressing these GCRs compared to GCRs mediated by single copy sequences. These results indicate that the mechanisms for formation and suppression of rearrangements occurring in regions containing “at risk” sequences differ from those occurring in regions of single copy sequence. This explains how extensive genome instability is prevented in eukaryotic cells whose genomes contain numerous divergent repeated sequences.

Rrm3 Protects the Saccharomyces cerevisiae Genome From Instability at Nascent Sites of Retrotransposition

The DNA helicase Rrm3 promotes replication fork progression through >1000 discrete genomic regions and represses the cDNA-mediated mobility of the Ty1 retrotransposon. We explored the connection between DNA replication and Ty1 retromobility by investigating the basis of increased retromobility in an rrm3 mutant. Even though Ty1 cDNA levels are increased in the absence of RRM3, neither the level nor target-site specificity of cDNA integration was altered. Instead, cDNA was incorporated into the genome by a Rad52-dependent mechanism that did not involve gene conversion of genomic Ty1 sequences. In rrm3 isolates, incorporated cDNA was often present in tandem arrays. Multimeric cDNA arrays probably arise during chromosomal break repair, since their appearance was strongly correlated with the formation of gross chromosomal rearrangements. Moreover, Ty1 multimers were invariantly located on rearranged chromosomes, when present. Overexpression of a cellular RNase H, which degrades RNA in an RNA:DNA hybrid, completely suppressed the increase in Ty1 multimer formation in an rrm3 mutant. We propose that RNA:DNA hybrid regions within nascent retrotransposition events block replication in an rrm3 mutant, leading to chromosome breaks within Ty1 sequences. Multiple extragenomic Ty1 cDNA molecules are then used as donors in recombinational repair of the break before it is healed.

Cdc28/Cdk1 positively and negatively affects genome stability in S. cerevisiae

We studied the function of the cyclin-dependent kinase Cdc28 (Cdk1) in the DNA damage response and maintenance of genome stability using Saccharomyces cerevisiae. Reduced Cdc28 activity sensitizes cells to chronic DNA damage, but Cdc28 is not required for cell viability upon acute exposure to DNA-damaging agents. Cdc28 is also not required for activation of the DNA damage and replication checkpoints. Chemical–genetic analysis reveals that CDC28 functions in an extensive network of pathways involved in maintenance of genome stability, including homologous recombination, sister chromatid cohesion, the spindle checkpoint, postreplication repair, and telomere maintenance. In addition, Cdc28 and Mre11 appear to cooperate to prevent mitotic catastrophe after DNA replication arrest. We show that reduced Cdc28 activity results in suppression of gross chromosomal rearrangements (GCRs), indicating that Cdc28 is required for formation or recovery of GCRs. Thus, we conclude that Cdc28 functions in a genetic network that supports cell viability during DNA damage while promoting the formation of GCRs.

Spt2p Defines a New Transcription-Dependent Gross Chromosomal Rearrangement Pathway

Large numbers of gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. High mobility group 1 (HMG1) protein is a non-histone DNA-binding protein and is highly expressed in different types of tumors. The high expression of HMG1 could alter DNA structure resulting in GCRs. Spt2p is a non-histone DNA binding protein in Saccharomyces cerevisiae and shares homology with mammalian HMG1 protein. We found that Spt2p overexpression enhances GCRs dependent on proteins for transcription elongation and polyadenylation. Excess Spt2p increases the number of cells in S phase and the amount of single-stranded DNA (ssDNA) that might be susceptible to cause DNA damage and GCR. Consistently, RNase H expression, which reduces levels of ssDNA, decreased GCRs in cells expressing high level of Spt2p. Lastly, high transcription in the chromosome V, the location at which GCR is monitored, also enhanced GCR formation. We propose a new pathway for GCR where DNA intermediates formed during transcription can lead to genomic instability.

A Screen for Suppressors of Gross Chromosomal Rearrangements Identifies a Conserved Role for PLP in Preventing DNA Lesions

Genome instability is a hallmark of cancer cells. One class of genome aberrations prevalent in tumor cells is termed gross chromosomal rearrangements (GCRs). GCRs comprise chromosome translocations, amplifications, inversions, deletion of whole chromosome arms, and interstitial deletions. Here, we report the results of a genome-wide screen in Saccharomyces cerevisiae aimed at identifying novel suppressors of GCR formation. The most potent novel GCR suppressor identified is BUD16, the gene coding for yeast pyridoxal kinase (Pdxk), a key enzyme in the metabolism of pyridoxal 5′ phosphate (PLP), the biologically active form of vitamin B6. We show that Pdxk potently suppresses GCR events by curtailing the appearance of DNA lesions during the cell cycle. We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint. Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase. Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

The absence of Tsa1, a key peroxiredoxin that functions to scavenge H(2)O(2) in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations including gross chromosomal rearrangements (GCRs). Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD6 and several key genes involved in DNA double-strand break repair. In the present study we investigated the causes of GCRs and cell death in these mutants. tsa1-associated GCRs were independent of the activity of the translesion DNA polymerases zeta, eta, and Rev1. Anaerobic growth reduced substantially GCR rates of WT and tsa1 mutants and restored the viability of tsa1 rad6, tsa1 rad51, and tsa1 mre11 double mutants. Anaerobic growth also reduced the GCR rate of rad27, pif1, and rad52 mutants, indicating a role of reactive oxygen species in GCR formation in these mutants. In addition, deletion of TSA1 or H(2)O(2) treatment of WT cells resulted in increased formation of Rad52 foci, sites of repair of multiple DNA lesions. H(2)O(2) treatment also induced the GCRs. Our results provide in vivo evidence that oxygen metabolism and reactive oxygen species are important sources of DNA damages that can lead to GCRs and lethal effects in S. cerevisiae.

Measuring the rate of gross chromosomal rearrangements in Saccharomyces cerevisiae: A practical approach to study genomic rearrangements observed in cancer

Gross chromosomal rearrangements (GCRs), including translocations, deletions, amplifications and aneuploidy are frequently observed in various types of human cancers. Despite their clear importance in carcinogenesis, the molecular mechanisms by which GCRs are generated and held in check are poorly understood. By using a GCR assay, which can measure the rate of accumulation of spontaneous GCRs in Saccharomyces cerevisiae, we have found that many proteins involved in DNA replication, DNA repair, DNA recombination, checkpoints, chromosome remodeling, and telomere maintenance, play crucial roles in GCR metabolism. We describe here the theoretical background and practical procedures of this GCR assay. We will explain the breakpoint structure and DNA damage that lead to GCR formation. We will also summarize the pathways that suppress and enhance GCR formation. Finally, we will briefly describe similar assays developed by others and discuss their potential in studying GCR metabolism.

Suppression of gross chromosomal rearrangements by yKu70-yKu80 heterodimer through DNA damage checkpoints

The inactivation of either subunit of the Ku70-Ku80 heterodimer, which functions in nonhomologous end-joining and telomere maintenance, generates severe defects such as sensitivity to DNA damage, telomere shortening, and increased gross chromosomal rearrangements (GCRs) that are frequently observed in many cancers. To understand the mechanism of Ku as a genome gatekeeper, we overexpressed the yKu70-yKu80 heterodimer and monitored the formation of GCRs. Ku overexpression suppressed the formation of either spontaneously generated GCRs or those induced by treatments with different DNA damaging agents. Interestingly, this suppression depended on Ku's interaction with DNA damage checkpoints and not through nonhomologous end-joining. We also demonstrate that the inactivation of telomerase inhibitor, Pif1 along with Ku overexpression or the overexpression of Pif1 in either yku70 or yku80 strains arrested the cell cycle at S phase in a DNA damage checkpoint-dependent fashion. Lastly, Ku overexpression causes cell growth delay, which depends on intact Rad27. In summary, the results presented here suggest that Ku functions as a genomic gatekeeper through its crosstalk with DNA damage checkpoints.

Mutation in Rpa1 results in defective DNA double-strand break repair, chromosomal instability and cancer in mice

Most cancers have multiple chromosomal rearrangements; the molecular mechanisms that generate them remain largely unknown. Mice carrying a heterozygous missense change in one of the DNA-binding domains of Rpa1 develop lymphoid tumors, and their homozygous littermates succumb to early embryonic lethality. Array comparative genomic hybridization of the tumors identified large-scale chromosomal changes as well as segmental gains and losses. The Rpa1 mutation resulted in defects in DNA double-strand break repair and precipitated chromosomal breaks as well as aneuploidy in primary heterozygous mutant mouse embryonic fibroblasts. The equivalent mutation in yeast is hypomorphic and semidominant and enhanced the formation of gross chromosomal rearrangements in multiple genetic backgrounds. These results indicate that Rpa1 functions in DNA metabolism are essential for the maintenance of chromosomal stability and tumor suppression.

Mitotic checkpoint function in the formation of gross chromosomal rearrangements in Saccharomyces cerevisiae.

The accumulation of gross chromosomal rearrangements (GCRs) is characteristic of cancer cells. Multiple pathways that prevent GCRs, including S-phase cell cycle checkpoints, homologous recombination, telomere maintenance, suppression of de novo telomere addition, chromatin assembly, and mismatch repair, have been identified in Saccharomyces cerevisiae. However, pathways that promote the formation of GCRs are not as well understood. Of these, the de novo telomere addition pathway and nonhomologous end-joining are the best characterized. Here, we demonstrate that defects in the mitotic checkpoint and the mitotic exit network can suppress GCRs in strains containing defects that increase the GCR rate. These data suggest that functional mitotic checkpoints can play a role in the formation of genome rearrangements.

Chromosome integrity in Saccharomyces cerevisiae: the interplay of DNA replication initiation factors, elongation factors, and origins.

The integrity of chromosomes during cell division is ensured by both trans-acting factors and cis-acting chromosomal sites. Failure of either these chromosome integrity determinants (CIDs) can cause chromosomes to be broken and subsequently misrepaired to form gross chromosomal rearrangements (GCRs). We developed a simple and rapid assay for GCRs, exploiting yeast artificial chromosomes (YACs) in Saccharomyces cerevisiae. We used this assay to screen a genome-wide pool of mutants for elevated rates of GCR. The analyses of these mutants define new CIDs (Orc3p, Orc5p, and Ycs4p) and new pathways required for chromosome integrity in DNA replication elongation (Dpb11p), DNA replication initiation (Orc3p and Orc5p), and mitotic condensation (Ycs4p). We show that the chromosome integrity function of Orc5p is associated with its ATP-binding motif and is distinct from its function in controlling the efficiency of initiation of DNA replication. Finally, we used our YAC assay to assess the interplay of trans and cis factors in chromosome integrity. Increasing the number of origins on a YAC suppresses GCR formation in our dpb11 mutant but enhances it in our orc mutants. This result provides potential insights into the counterbalancing selective pressures necessary for the evolution of origin density on chromosomes.

Induction of genome instability by DNA damage in Saccharomyces cerevisiae

The accumulation of gross chromosomal rearrangements (GCRs) is a characteristic of many types of cancer cells, although it is unclear what defects cause these rearrangements and how the different types of GCRs observed are formed. In the present study, we have used a Saccharomyces cerevisiae system for measuring GCRs to analyze the ability of a variety of DNA damaging agents to induce GCRs. The two most potent inducers of GCRs observed were methyl methane sulfonate (MMS) and HO-endonuclease-induced double strand breaks (DSBs). Bleomycin, camptothecan and γ-irradiation induced intermediate levels of GCRs and cisplatin induced very low levels of GCRs whereas N-methyl- N′ -nitro- N -nitrosoguanidine (MNNG) and ethyl methane sulfonate (EMS) primarily induced base substitution mutations. MMS treatment primarily induced rearrangements in which the end of a chromosome was deleted and a new telomere was added (telomere additions) and also induced translocations. Consistent with this GCR spectrum, the formation of MMS-induced GCRs was primarily dependent on telomere maintenance functions and were completely eliminated in mutants that were defective for both telomere maintenance functions and non-homologous end joining (NHEJ). In contrast, HO-endonuclease DSBs induced mostly translocations and interstitial deletions whereas few telomere additions were observed. Genetic analysis indicated that HO DSB-induced GCRs were suppressed by a number of pathways including the DNA damage checkpoints, DSB repair pathways and NHEJ.

Gross chromosomal rearrangements and genetic exchange between nonhomologous chromosomes following BRCA2 inactivation

Cancer-causing mutations often arise from gross chromosomal rearrangements (GCRs) such as translocations, which involve genetic exchange between nonhomologous chromosomes. Here we show that murine Brca2 has an essential function in suppressing GCR formation after chromosome breakage. Cells that harbor truncated Brca2 spontaneously incur GCRs and genomic DNA breaks during division. They exhibit hypersensitivity to DNA damage by interstrand cross-linkers, which even at low doses trigger aberrant genetic exchange between nonhomologous chromosomes. Therefore, genetic instability in Brca2-deficient cells results from the mutagenic processing of spontaneous or induced DNA damage into gross chromosomal rearrangements, providing a mechanistic basis for cancer predisposition.

Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants.

Cancer progression is often associated with the accumulation of gross chromosomal rearrangements (GCRs), such as translocations, deletion of a chromosome arm, interstitial deletions or inversions. In many instances, GCRs inactivate tumour-suppressor genes or generate novel fusion proteins that initiate carcinogenesis. The mechanism underlying GCR formation appears to involve interactions between DNA sequences of little or no homology. We previously demonstrated that mutations in the gene encoding the largest subunit of the Saccharomyces cerevisiae single-stranded DNA binding protein (RFA1) increase microhomology-mediated GCR formation. To further our understanding of GCR formation, we have developed a novel mutator assay in S. cerevisiae that allows specific detection of such events. In this assay, the rate of GCR formation was increased 600-5, 000-fold by mutations in RFA1, RAD27, MRE11, XRS2 and RAD50, but was minimally affected by mutations in RAD51, RAD54, RAD57, YKU70, YKU80, LIG4 and POL30. Genetic analysis of these mutants suggested that at least three distinct pathways can suppress GCRs: two that suppress microhomology-mediated GCRs (RFA1 and RAD27) and one that suppresses non-homology-mediated GCRs (RAD50/MRE11/XRS2).