Extensive loss of heterozygosity is suppressed during homologous repair of chromosomal breaks

Loss of heterozygosity (LOH) is a common genetic alteration in tumors and often extends several megabases to encompass multiple genetic loci or even whole chromosome arms. Based on marker and karyotype analysis of tumor samples, a significant fraction of LOH events appears to arise from mitotic recombination between homologous chromosomes, reminiscent of recombination during meiosis. As DNA double-strand breaks (DSBs) initiate meiotic recombination, a potential mechanism leading to LOH in mitotically dividing cells is DSB repair involving homologous chromosomes. We therefore sought to characterize the extent of LOH arising from DSB-induced recombination between homologous chromosomes in mammalian cells. To this end, a recombination reporter was introduced into a mouse embryonic stem cell line that has nonisogenic maternal and paternal chromosomes, as is the case in human populations, and then a DSB was introduced into one of the chromosomes. Recombinants involving alleles on homologous chromosomes were readily obtained at a frequency of 4.6 x 10(-5); however, this frequency was substantially lower than that of DSB repair by nonhomologous end joining or the inferred frequency of homologous repair involving sister chromatids. Strikingly, the majority of recombinants had LOH restricted to the site of the DSB, with a minor class of recombinants having LOH that extended to markers 6 kb from the DSB. Furthermore, we found no evidence of LOH extending to markers 1 centimorgan or more from the DSB. In addition, crossing over, which can lead to LOH of a whole chromosome arm, was not observed, implying that there are key differences between mitotic and meiotic recombination mechanisms. These results indicate that extensive LOH is normally suppressed during DSB-induced allelic recombination in dividing mammalian cells.     

Illegitimate recombination leading to allelic loss and unbalanced translocation in p53-mutated human lymphoblastoid cells.

Allelic loss and translocation are critical mutational events in human tumorigenesis. Allelic loss, which is usually identified as loss of heterozygosity (LOH), is frequently observed at tumor suppressor loci in various kinds of human tumors. It is generally thought to result from deletion or mitotic recombination between homologous chromosomes. In this report, we demonstrate that illegitimate (nonhomologous) recombination strongly contributes to the generation of allelic loss in p53-mutated cells. Spontaneous and X-ray-induced LOH mutations at the heterozygous thymidine kinase (tk) gene, which is located on the long arm of chromosome 17, from normal (TK6) and p53-mutated (WTK-1) human lymphoblastoid cells were cytogenetically analyzed by chromosome 17 painting. We observed unbalanced translocations in 53% of LOH mutants spontaneously arising from WTK-1 cells but none spontaneously arising from TK6 cells. We postulate that illegitimate recombination was occurring between nonhomologous chromosomes after DNA replication, leading to allelic loss and unbalanced translocations in p53-mutated WTK-1 cells. X-ray irradiation, which induces DNA double-strand breaks (DSBs), enhanced the generation of unbalanced translocation more efficiently in WTK-1 than in TK6 cells. This observation implicates the wild-type p53 protein in the regulation of homologous recombination and recombinational DNA repair of DSBs and suggests a possible mechanism by which loss of p53 function may cause genomic instability.     

Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.     

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle

A CAN1/can1Δ heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.     

Spontaneous loss of heterozygosity in diploid Saccharomyces cerevisiae cells

To obtain a broad perspective of the events leading to spontaneous loss of heterozygosity (LOH), we have characterized the genetic alterations that functionally inactivated the URA3 marker hemizygously or heterozygously situated either on chromosome III or chromosome V in diploid Saccharomyces cerevisiae cells. Analysis of chromosome structure in a large number of LOH clones by pulsed-field gel electrophoresis and PCR showed that chromosome loss, allelic recombination, and chromosome aberration were the major classes of genetic alterations leading to LOH. The frequencies of chromosome loss and chromosome aberration were significantly affected when the marker was located in different chromosomes, suggesting that chromosome-specific elements may affect the processes that led to these alterations. Aberrant-sized chromosomes were detected readily in approximately 8% of LOH events when the URA3 marker was placed in chromosome III. Molecular mechanisms underlying the chromosome aberrations were further investigated by studying the fate of two other genetic markers on chromosome III. Chromosome aberration caused by intrachromosomal rearrangements was predominantly due to a deletion between the MAT and HMR loci that occurred at a frequency of 3.1 x 10(-6). Another type of chromosome aberration, which occurred at a frequency slightly higher than that of the intrachromosomal deletion, appeared to be caused by interchromosomal rearrangement, including unequal crossing over between homologous chromatids and translocation with another chromosome.     

Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.     

Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast

Pairing of homologous chromosomes is important for homologous recombination and correct chromosome segregation during meiosis. It has been proposed that telomere clustering, nuclear oscillation, and recombination during meiotic prophase facilitate homologous chromosome pairing in fission yeast. Here we examined the contributions of these chromosomal events to homologous chromosome pairing, by directly observing the dynamics of chromosomal loci in living cells of fission yeast. Homologous loci exhibited a dynamic process of association and dissociation during the time course of meiotic prophase. Lack of nuclear oscillation reduced association frequency for both centromeric and arm regions of the chromosome. Lack of telomere clustering or recombination reduced association frequency at arm regions, but not significantly at centromeric regions. Our results indicate that homologous chromosomes are spatially aligned by oscillation of telomere-bundled chromosomes and physically linked by recombination at chromosome arm regions; this recombination is not required for association of homologous centromeres.     

Chromosomal Translocations Generated by High-Frequency Meiotic Recombination between Repeated Yeast Genes

We have examined meiotic and mitotic recombination between repeated genes on nonhomologous chromosomes in the yeast Saccharomyces cerevisiae. The results of these experiments can be summarized in three statements. First, gene conversion events between repeats on nonhomologous chromosomes occur frequently in meiosis. The frequency of such conversion events is only 17-fold less than the analogous frequency of conversion between genes at allelic positions on homologous chromosomes. Second, meiotic and mitotic conversion events between repeated genes on nonhomologous chromosomes are associated with reciprocal recombination to the same extent as conversion between allelic sequences. The reciprocal exchanges between the repeated genes result in chromosomal translocations. Finally, recombination between repeated genes on nonhomologous chromosomes occurs much more frequently in meiosis than in mitosis.     

Accurate homologous recombination is a prominent double-strand break repair pathway in mammalian chromosomes and is modulated by mismatch repair protein Msh2

We designed DNA substrates to study intrachromosomal recombination in mammalian chromosomes. Each substrate contains a thymidine kinase (tk) gene fused to a neomycin resistance (neo) gene. The fusion gene is disrupted by an oligonucleotide containing the 18-bp recognition site for endonuclease I-SceI. Substrates also contain a “donor” tk sequence that displays 1% or 19% sequence divergence relative to the tk portion of the fusion gene. Each donor serves as a potential recombination partner for the fusion gene. After stably transfecting substrates into mammalian cell lines, we investigated spontaneous recombination and double-strand break (DSB)-induced recombination following I-SceI expression. No recombination events between sequences with 19% divergence were recovered. Strikingly, even though no selection for accurate repair was imposed, accurate conservative homologous recombination was the predominant DSB repair event recovered from rodent and human cell lines transfected with the substrate containing sequences displaying 1% divergence. Our work is the first unequivocal demonstration that homologous recombination can serve as a major DSB repair pathway in mammalian chromosomes. We also found that Msh2 can modulate homologous recombination in that Msh2 deficiency promoted discontinuity and increased length of gene conversion tracts and brought about a severalfold increase in the overall frequency of DSB-induced recombination.     

High frequency induction of mitotic recombination by ionizing radiation in Mlh1 null mouse cells

Mitotic recombination in somatic cells involves crossover events between homologous autosomal chromosomes. This process can convert a cell with a heterozygous deficiency to one with a homozygous deficiency if a mutant allele is present on one of the two homologous autosomes. Thus mitotic recombination often represents the second mutational step in tumor suppressor gene inactivation. In this study we examined the frequency and spectrum of ionizing radiation (IR)-induced autosomal mutations affecting Aprt expression in a mouse kidney cell line null for the Mlh1 mismatch repair (MMR) gene. The mutant frequency results demonstrated high frequency induction of mutations by IR exposure and the spectral analysis revealed that most of this response was due to the induction of mitotic recombinational events. High frequency induction of mitotic recombination was not observed in a DNA repair-proficient cell line or in a cell line with an MMR-independent mutator phenotype. These results demonstrate that IR exposure can initiate a process leading to mitotic recombinational events and that MMR function suppresses these events from occurring.     

Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae

In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements.     

Analysis of spontaneous gene conversion tracts within and between mammalian chromosomes

In the present study, we report the first characterization of gene conversion tract length, continuity and fidelity for pathways of gene targeting, ectopic and intrachromosomal homologous recombination using the same locus and mammalian somatic cell type. In this isogenic cell system, the vast majority of recombinants (> 97%) are generated by homologous recombination and display a high degree of fidelity in the gene conversion process. Individual gene conversion tracts are highly likely to involve single, independent recombination events and proceed through a heteroduplex DNA intermediate. In all recombination pathways, gene conversion tracts are long, extending up to ∼ 2 kb. Most gene conversion tracts are continuous in favor of donor region sequences, but in a small fraction of recombinants (15%), discontinuous gene conversion tracts are observed. In most cases, the recombination donor sequence is unaltered, although in two cases of intrachromosomal recombination, both recombination donor and recipient sequences bear gene conversion tracts. Overall, gene conversion events are similar, both qualitatively and quantitatively, for homologous recombination within and between mammalian chromosomes.     

Mutagenic radioadaptation in a human lymphoblastoid cell line

We investigated the mutagenic radioadaptive response of human lymphoblastoid TK6 cells by pretreating them with a low dose (5 cGy) of X-rays followed by a high (2 Gy) dose 6h later. Pretreatment reduced the 2-Gy-induced mutation frequency (MF) of the thymidine kinase (TK) gene (18.3 x 10(-6)) to 62% of the original level (11.4 x 10(-6)). A loss of heterozygosity (LOH) detection analysis applied to the isolated TK(-) mutants revealed the mutational events as non-LOH (resulting mostly from a point mutation in the TK gene), hemizygous LOH (resulting from a chromosomal deletion), or homozygous LOH (resulting from homologous recombination (HR) between chromosomes). For non-LOH events, pretreatment decreased the frequency to 27% of the original level (from 7.1 x 10(-6) to 1.9 x 10(-6)). cDNAs prepared from the non-LOH mutants revealed that the decrease was due mainly to the repression of base substitutions. The frequency of hemizygous LOH events, however, was not significantly altered by pretreatment. Mapping analysis of chromosome 17 demonstrated that the distribution and the extent of hemizygous LOH events were also not significantly influenced by pretreatment. For homozygous LOH events, pretreatment reduced the frequency to 61% of the original level (from 5.1 x 10(-6) to 3.1 x 10(-6)), reflecting an enhancement in HR repair of DNA double-strand breaks. Our findings suggest that the radioadaptive response in TK6 cells follows mainly from mutations at the base-sequence level, not the chromosome level.     

Elevated incidence of loss of heterozygosity (LOH) in an sgs1 mutant of Saccharomyces cerevisiae: roles of yeast RecQ helicase in suppression of aneuploidy,interchromosomal rearrangement,and the simultaneous incidence of both events during mitotic growth

The SGS1 gene of Saccharomyces cerevisiae is a member of the RecQ helicase family, which includes the human BLM, WRN and RECQL4 genes responsible for Bloom and Werner's syndrome and Rothmund-Thomson syndrome, respectively. Cells defective in any of these genes exhibit a higher incidence of genome instability. We previously demonstrated that various genetic alterations were detectable as events leading to loss of heterozygosity (LOH) in S. cerevisiae diploid cells, utilizing a hemizygous URA3 marker placed at the center of the right arm of chromosome III. Analyses of chromosome structure in LOH clones by pulse field gel electrophoresis (PFGE) and PCR, coupled with a genetic method, allow identification of genetic alterations leading to the LOH. Such alterations include chromosome loss, chromosomal rearrangements at various locations and intragenic mutation. In this work, we have investigated the LOH events occurring in cells lacking the SGS1 gene. The frequencies of all types of LOH events, excluding intragenic mutation, were increased in sgs1 null mutants as compared to the wild-type cells. Loss of chromosome III and chromosomal rearrangements were increased 13- and 17-fold, respectively. Further classification of the chromosomal rearrangements confirmed that two kinds of events were especially increased in the sgs1 mutants: (1) ectopic recombination between chromosomes, that is, unequal crossing over and translocation (46-fold); and (2) allelic crossing over associated with chromosome loss (40-fold). These findings raise the possibility that the Sgs1 protein is involved in the processing of recombination intermediates as well as in the prevention of recombination repair during chromosome DNA replication. On the other hand, intrachromosomal deletions between MAT and HMR were increased only slightly (2.9-fold) in the sgs1 mutants. These results clearly indicate that defects in the SGS1 gene function lead to an elevated incidence of LOH in multiple ways, including chromosome loss and interchromosomal rearrangements, but not intrachromosomal deletion.     

Spontaneous chromosome loss in Saccharomyces cerevisiae is suppressed by DNA damage checkpoint functions.

Genomic instability is one of the hallmarks of cancer cells and is often the causative factor in revealing recessive gene mutations that progress cells along the pathway to unregulated growth. Genomic instability can take many forms, including aneuploidy and changes in chromosome structure. Chromosome loss, loss and reduplication, and deletions are the majority events that result in loss of heterozygosity (LOH). Defective DNA replication, repair, and recombination can significantly increase the frequency of spontaneous genomic instability. Recently, DNA damage checkpoint functions that operate during the S-phase checkpoint have been shown to suppress spontaneous chromosome rearrangements in haploid yeast strains. To further study the role of DNA damage checkpoint functions in genomic stability, we have determined chromosome loss in DNA damage checkpoint-deficient yeast strains. We have found that the DNA damage checkpoints are essential for preserving the normal chromosome number and act synergistically with homologous recombination functions to ensure that chromosomes are segregated correctly to daughter cells. Failure of either of these processes increases LOH events. However, loss of the G2/M checkpoint does not result in an increase in chromosome loss, suggesting that it is the various S-phase DNA damage checkpoints that suppress chromosome loss. The mec1 checkpoint function mutant, defective in the yeast ATR homolog, results in increased recombination through a process that is distinct from that operative in wild-type cells.     

Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants

Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10–60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, “secondary” recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Homologous recombination via synthesis-dependent strand annealing in yeast requires the Irc20 and Srs2 DNA helicases

Synthesis-dependent strand-annealing (SDSA)-mediated homologous recombination replaces the sequence around a DNA double-strand break (DSB) with a copy of a homologous DNA template, while maintaining the original configuration of the flanking regions. In somatic cells at the 4n stage, Holliday-junction-mediated homologous recombination and nonhomologous end joining (NHEJ) cause crossovers (CO) between homologous chromosomes and deletions, respectively, resulting in loss of heterozygosity (LOH) upon cell division. However, the SDSA pathway prevents DSB-induced LOH. We developed a novel yeast DSB-repair assay with two discontinuous templates, set on different chromosomes, to determine the genetic requirements for somatic SDSA and precise end joining. At first we used our in vivo assay to verify that the Srs2 helicase promotes SDSA and prevents imprecise end joining. Genetic analyses indicated that a new DNA/RNA helicase gene, IRC20, is in the SDSA pathway involving SRS2. An irc20 knockout inhibited both SDSA and CO and suppressed the srs2 knockout-induced crossover enhancement, the mre11 knockout-induced inhibition of SDSA, CO, and NHEJ, and the mre11-induced hypersensitivities to DNA scissions. We propose that Irc20 and Mre11 functionally interact in the early steps of DSB repair and that Srs2 acts on the D-loops to lead to SDSA and to prevent crossoverv.     

The Red Queen theory of recombination hotspots

Recombination hotspots are small chromosomal regions, where meiotic crossover events happen with high frequency. Recombination is initiated by a double‐strand break (DSB) that requires the intervention of the molecular repair mechanism. The DSB repair mechanism may result in the exchange of homologous chromosomes (crossover) and the conversion of the allelic sequence that breaks into the one that does not break (biased gene conversion). Biased gene conversion results in a transmission advantage for the allele that does not break, thus preventing recombination and rendering recombination hotspots transient. How is it possible that recombination hotspots persist over evolutionary time (maintaining the average chromosomal crossover rate) when they are self‐destructive? This fundamental question is known as the recombination hotspot paradox and has attracted much attention in recent years. Yet, that attention has not translated into a fully satisfactory answer. No existing model adequately explains all aspects of the recombination hotspot paradox. Here, we formulate an intragenomic conflict model resulting in Red Queen dynamics that fully accounts for all empirical observations regarding the molecular mechanisms of recombination hotspots, the nonrandom targeting of the recombination machinery to hotspots and the evolutionary dynamics of hotspot turnover.     

The Efficiency of Meiotic Recombination between Dispersed Sequences in Saccharomyces Cerevisiae Depends upon Their Chromosomal Location

To examine constraints imposed on meiotic recombination by homologue pairing, we measured the frequency of recombination between mutant alleles of the ARG4 gene contained in pBR322-based inserts. Inserts were located at identical loci on homologues (allelic recombination) or at different loci on either homologous or heterologous chromosomes (ectopic recombination). Ectopic recombination between interstitially located inserts on heterologous chromosomes had an efficiency of 6-12% compared to allelic recombination. By contrast, ectopic recombination between interstitial inserts located on homologues had relative efficiencies of 47-99%. These findings suggest that when meiotic ectopic recombination occurs, homologous chromosomes are already colocalized. The efficiency of ectopic recombination between inserts on homologues decreased as the physical distance between insert sites was increased. This result is consistent with the suggestion that during meiotic recombination, homologues are not only close to each other, but also are aligned end to end. Finally, the efficiency of ectopic recombination between inserts near telomeres (within 16 kb) was significantly greater than that observed with inserts >50 kb from the nearest telomere. Thus, at the time of recombination, there may be a special relationship between the ends of chromosomes not shared with interstitial regions.