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.     

Conjugational Recombination in Escherichia Coli: Genetic Analysis of Recombinant Formation in Hfr X F(-) Crosses

The formation of recombinants during conjugation between Hfr and F(-) strains of Escherichia coli was investigated using unselected markers to monitor integration of Hfr DNA into the circular recipient chromosome. In crosses selecting a marker located ~500 kb from the Hfr origin, 60-70% of the recombinants appeared to inherit the Hfr DNA in a single segment, with the proximal exchange located >300 kb from the selected marker. The proportion of recombinants showing multiple exchanges increased in matings selecting more distal markers located 700-2200 kb from the origin, but they were always in the minority. This effect was associated with decreased linkage of unselected proximal markers. Mutation of recB, or recD plus recJ, in the recipient reduced the efficiency of recombination and shifted the location of the proximal exchange (s) closer to the selected marker. Mutation of recF, recO or recQ produced recombinants in which this exchange tended to be closer to the origin, though the effect observed was rather small. Up to 25% of recombinant colonies in rec(+) crosses showed segregation of both donor and recipient alleles at a proximal unselected locus. Their frequency varied with the distance between the selected and unselected markers and was also related directly to the efficiency of recombination. Mutation of recD increased their number by twofold in certain crosses to a value of 19%, a feature associated with an increase in the survival of linear DNA in the absence of RecBCD exonuclease. Mutation of recN reduced sectored recombinants in these crosses to ~1% in all the strains examined, including recD. A model for conjugational recombination is proposed in which recombinant chromosomes are formed initially by two exchanges that integrate a single piece of duplex Hfr DNA into the recipient chromosome. Additional pairs of exchanges involving the excised recipient DNA, RecBCD enzyme and RecN protein, can subsequently modify the initial product to generate the spectrum of recombinants normally observed.     

Expression of Saccharomyces cerevisiae MATa and MAT alpha enhances the HO endonuclease-stimulation of chromosomal rearrangements directed by his3 recombinational substrates

Radiation resistance in Saccharomyces cerevisiae is greater in a/alpha diploids than in aa or alpha alpha diploids, and higher levels of radiation resistance correlates with more mitotic recombination. Specifically, we investigated whether the stimulation of directed translocations, inversions, and unequal sister chromatid exchanges (SCEs) by HO endonuclease-induced double-strand breaks (DSBs) is enhanced in a/alpha cells. These rearrangements result from mitotic recombination between two truncated his3 genes, his3-delta 5' and his3-delta 3'::HOcs, positioned on non-homologous chromosomes or positioned in juxtaposition on the same chromosome in inverted or direct orientation. Mitotic recombination was initiated by HO endonuclease-induced DSBs at the HO cut site (HOcs) located at his3-delta 3'::HOcs, and His+ recombinants were selected. In MATa-inc haploid strains, which do not switch mating-type, the DSB reduced viability, relative to undamaged cells, and increases the frequency of His+ recombinants containing translocations to 2.4 x 10(-4) (seven-fold), SCEs to 5.4 x 10(-4) (five-fold), and inversions to 1.8 x 10(-3) (six-fold). Compared to a haploids, DSB-stimulated frequencies in a/alpha haploids were three-fold higher for translocations, two-fold higher for SCEs, and ten-fold higher for inversions; however DSB-induced lethality was greater in a/alpha haploids. Compared to aa diploids, DSB-stimulated frequencies of translocations and viability after chromosome cleavage were greater in a/alpha diploids. We suggest that heterozygosity at MAT may elevate the frequency of DSB-initiated reciprocal exchange events in both haploid and diploid cells, but may only increase viability after chromosome cleavage in diploid cells.     

Characterisation of wheat-rye recombinants with RFLP and PCR probes

Summary The introgression of genetic material from alien species into wheat has become an important tool in modern wheat breeding. Ideally, only the trait of interest and no flanking material should be transferred. Random recombination between the genetic material is therefore of paramount importance. In a model system, we examined 17 recombinants putatively between chromosome 1D of wheat and 1R of rye with 60 random RFLP and three PCR markers. The recombinants had been generated by removing the normal effect of the Ph1 gene in the wheat background. Amongst the nine short-arm recombinants, three breakpoints were identified but no differentiation could be made between the five proximal recombinants. For the eight long-arm recombinants analysed only two breakpoints were identified with 36 markers. However, only a single RFLP marker was able to differentiate between the recombinants. Indeed the long-arm results are consistent with the possibility that only the rye telomeric region had been transferred. These results indicate either a strong clustering of the RFLP markers near the centromere or else imply that recombination induced between wheat and rye in the absence of the normal effect of the Ph1 gene occurs at only restricted sites. The results allow new primary recombinants to be selected for intercrossing to generate secondary recombinants which are expected to have a smaller interstitial rye segment than that present in DR-A1.     

UV stimulation of chromosomal marker exchange in Sulfolobus acidocaldarius: implications for DNA repair, conjugation and homologous recombination at extremely high temperatures

The hyperthermophilic archaeon Sulfolobus acidocaldarius exchanges and recombines chromosomal markers by a conjugational mechanism, and the overall yield of recombinants is greatly increased by previous exposure to UV light. This stimulation was studied in an effort to clarify its mechanism and that of marker exchange itself. A variety of experiments failed to identify a significant effect of UV irradiation on the frequency of cell pairing, indicating that subsequent steps are primarily affected, i.e., transfer of DNA between cells or homologous recombination. The UV-induced stimulation decayed rather quickly in parental cells during preincubation at 75 degrees, and the rate of decay depended on the incubation temperature. Preincubation at 75 degrees decreased the yield of recombinants neither from unirradiated parental cells nor from parental suspensions subsequently irradiated. We interpret these results as evidence that marker exchange is stimulated by recombinogenic DNA lesions formed as intermediates in the process of repairing UV photoproducts in the S. acidocaldarius chromosome.     

UV irradiation causes the loss of viable mitotic recombinants in Schizosaccharomyces pombe cells lacking the G(2)/M DNA damage checkpoint

Elevated mitotic recombination and cell cycle delays are two of the cellular responses to UV-induced DNA damage. Cell cycle delays in response to DNA damage are mediated via checkpoint proteins. Two distinct DNA damage checkpoints have been characterized in Schizosaccharomyces pombe: an intra-S-phase checkpoint slows replication and a G(2)/M checkpoint stops cells passing from G(2) into mitosis. In this study we have sought to determine whether UV damage-induced mitotic intrachromosomal recombination relies on damage-induced cell cycle delays. The spontaneous and UV-induced recombination phenotypes were determined for checkpoint mutants lacking the intra-S and/or the G(2)/M checkpoint. Spontaneous mitotic recombinants are thought to arise due to endogenous DNA damage and/or intrinsic stalling of replication forks. Cells lacking only the intra-S checkpoint exhibited no UV-induced increase in the frequency of recombinants above spontaneous levels. Mutants lacking the G(2)/M checkpoint exhibited a novel phenotype; following UV irradiation the recombinant frequency fell below the frequency of spontaneous recombinants. This implies that, as well as UV-induced recombinants, spontaneous recombinants are also lost in G(2)/M mutants after UV irradiation. Therefore, as well as lack of time for DNA repair, loss of spontaneous and damage-induced recombinants also contributes to cell death in UV-irradiated G(2)/M checkpoint mutants.     

Aspects of genetic interaction in hybrids of Aspergillus nidulans and Aspergillus rugulosus obtained by protoplast fusion

Hybrids were produced by protoplast fusion between strains of Aspergillus rugulosus and mitotic master strains of Aspergillus nidulans with a genetic marker on each linkage group. Analysis of segregants induced by growth on benomyl revealed recombination between every pair of unlinked markers. Parental combinations of markers were often recovered at significantly higher frequencies than expected. This aberrant segregation was not correlated with any particular pair of linkage-groups and was attributed to inter-species incompatibility. The segregation of genetic markers of A. rugulosus from the hybrids suggested that A. nidulans and A. rugulosus may differ in haploid chromosome number and chromosome size. In sexual crosses between A. nidulans and strains containing chromosomes of mixed parental origin recombinants were recovered. The results support the classification of A. nidulans and A. rugulosus as separate species.     

Mitotic recombination in cleavage divisions of Drosophila melanogaster. IV. Effect induced by radiation in female parents

The premutational changes induced by X-irradiation (3 kr or 1 kr) in X-chromosomes of female gametes gave rise to mitotic recombination between irradiated female X-chromosomes and non-irradiated male X-chromosomes in early cleavage nuclei of F1 daughters. Most of the recombinants were non-mosaic females. It can be concluded from the analysis of their phenotypes as well as their offsprings that all nuclei of somatic and germinal tissues of these females were descendants of one from the four first cleavage nuclei (the recombinant one). There were differences in rates and in patterns of recombination between experiments with different stocks. The recombination induced by treatment of female parents usually occurred in the proximal part of X-chromosomes.     

Meiosis Can Induce Recombination in rad52 Mutants of SACCHAROMYCES CEREVISIAE

The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks.     

Induced variations in microorganisms

Genetic transfer of markers has been found to occur through a sexual cycle between intact cells in a mixed culture of two different genotypes. When rS (radiosensitive and streptomycin resistant) and Rs (radioresistant and streptomycin sensitive) cells ofRhizobium trifolii were brought into varying periods of cellular contact, the cell mixture yielded RS recombinants. The recombination frequency was higher in young mating cultures than in aged ones. Except for the acquisition of the R and S markers, the recombinants did not differ appreciably from the parental strains in other characters.     

Mosaic analysis with double markers in mice

We describe a method termed MADM (mosaic analysis with double markers) in mice that allows simultaneous labeling and gene knockout in clones of somatic cells or isolated single cells in vivo. Two reciprocally chimeric genes, each containing the N terminus of one marker and the C terminus of the other marker interrupted by a loxP-containing intron, are knocked in at identical locations on homologous chromosomes. Functional expression of markers requires Cre-mediated interchromosomal recombination. MADM reveals that interchromosomal recombination can be induced efficiently in vivo in both mitotic and postmitotic cells in all tissues examined. It can be used to create conditional knockouts in small populations of labeled cells, to determine cell lineage, and to trace neuronal connections. To illustrate the utility of MADM, we show that cerebellar granule cell progenitors are fated at an early stage to produce granule cells with axonal projections limited to specific sublayers of the cerebellar cortex.     

The use of cell culture for subchromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.

Barley yellow dwarf virus (BYDV) resistance has been transferred to wheat from a group 7 chromosome of Thinopyrum (Agropyron) intermedium. The source of the resistance gene was the L1 disomic addition line, which carries the 7Ai-1 chromosome. The resistance locus is on the long arm of this chromosome. BYDV resistant recombinant lines were identified after three or more generations of selection against a group 7 Th. intermedium short arm marker (red coleoptile) and selection for the presence of BYDV resistance. One recombinant line produced by ph. mutant induced homoeologous pairing and 14 recombinant lines induced by cell culture have been identified. Resistance in seven of the cell culture induced recombinants has been inherited via pollen according to Mendelian segregation ratios for up to eight generations. Meiotic analysis of heterozygotes indicates that the alien chromatin in the cell culture induced recombinants is small enough to allow regular meiotic behaviour. The ph-induced recombinant was less regular in meiosis. A probe, pEleAcc2, originally isolated from Th. elongatum and that hybridizes to dispersed repeated DNA sequences, was utilised to detect Th. intermedium chromatin, which confers resistance to BYDV, in wheat backgrounds. Quantification of these hybridization signals indicated that the translocations involved a portion of alien chromatin that was smaller than the complete long arm of 7Ai-1. Restriction fragment length polymorphism analysis confirmed the loss of the short arm of 7Ai-1 and indicated the retention of segments of the long arm of 7Ai-1. Two 7Ai-1L DNA markers always assorted with the BYDV resistance. A third 7Ai-IL DNA marker was also present in seven of eight recombinants. In all recombinants except TC7, the 7Ai-1L markers replaced the 7DL markers. None of the wheat group 7 markers was missing from TC7. It is concluded that all the resistant lines are the result of recombination with wheat chromosome 7D, except line TC7, which is the result of recombination with an unidentified nongroup 7 chromosome.     

Separation of Linked Markers in Chinese Hamster Cell Hybrids: Mitotic Recombination Is Not Involved

A search for mitotic recombination was carried out using mutant subclones of cultured Chinese hamster ovary cells. Recombination events were sought between the linked loci specifying the enzymes hypoxanthine phosphoribosyl transferase and glucose-6-phosphate dehydrogenase. It was shown by fluctuation analysis that markers at these two loci co-segregate from doubly heterozygous pseudotetraploid hybrid cells more than 90% of the time. The minority class of segregants, which had lost one marker without losing the other, were genetically analyzed to distinguish between the possibilities of mitotic recombination and deletion of chromosomal material. Nine clones in which a linkage disruption had occured were studied, using further cell hybridization and segregation. In three cases, a recessive lethal loss of genetic information was indicated, suggesting the deletion mechanism. In six cases, it was demonstrated that no new linkage relationships had been established concomitant with linkage disruption. Thus, in all nine clones, the evidence indicated that mitotic recombination was not involved in the events that disrupted linkage between these two loci. If mitotic recombination takes place at all in this system, the rate must be less than about 10^-6 per cell per generation.     

The effect of three rad genes on survival, inter- and intragenic mitotic recombination in Saccharomyces. I. UV irradiation without photoreactivation or liquid-holding post-treatment

The effect of UV irradiation on the survival, inter- and intragenic mitotic recombination of 3 diploid UV sensitive Saccharomyces mutants was studied and compared with the wild type RAD. These strains, homozygous for either the RAD, r1s rad 9-4, or rad 2-20 gene, have DRF values for survival of 1:1.6:3:20.6 respectively, at LD1. Their recombination behaviour is not correlated to their survival characteristics. The RAD, r1s, and rad 2-20 strains showed UV induced mitotic inter- and intragenic recombinants; the induction in the r1s diploid is ca. 100 times greater for both the inter- and intragenic recombinants than in the RAD strain. The rad 9-4 diploid produced no UV induced mitotic recombinants whatsoever, and is therefore considered to be a rec- mutation.     

Genetic Analysis of Mouse T Haplotypes Using Mutations Induced by Ethylnitrosourea Mutagenesis: The Order of T and Qk Is Inverted in T Mutants

The t region of mouse chromosome 17 exhibits recombination suppression with wild-type chromatin. However, the region has resisted classical genetic dissection because of a lack of defined variants. Mutations induced by N-ethyl-N-nitrosourea (ENU) at the Brachyury (T), quaking (qk), and tufted (tf) loci of the mouse tw5 haplotype have now allowed the analysis of crossovers between two complete t haplotypes. A classical breeding analysis of the complete t haplotypes, tw5 and t12, utilizing the newly induced markers, reveals two inversions in t chromatin: one involving T and qk, and one involving tf and the H-2 complex. Moreover, the recombination frequency between the loci of T and qk is reduced compared to the frequency reported in normal chromatin. These two inversions are a sufficient explanation for the recombination inhibition with normal chromatin exhibited by t haplotypes isolated from the wild. Furthermore, the reduced recombination frequency between T and qk may indicate that the proximal gene rearrangement is not a simple inversion.     

Exchange of genetic markers at extremely high temperatures in the archaeon Sulfolobus acidocaldarius.

When cells of two auxotrophic mutants of Sulfolobus acidocaldarius are mixed and incubated on solid medium, they form stable genetic recombinants which can be selected, enumerated, and characterized. Any of a variety of auxotrophic markers can recombine with each other, and the phenomenon has been observed at temperatures of up to 84 degrees C. The ability to exchange and recombine chromosomal markers appears to be an intrinsic property of S. acidocaldarius strains. It occurs between two cell lines derived from the same parent or from different parents and also between a recombinant and its parent. This is the first observation of chromosomal marker exchange in archaea from geothermal environments and provides the first functional evidence of generalized, homologous recombination at such high temperatures.     

Evidence for biased holliday junction cleavage and mismatch repair directed by junction cuts during double-strand-break repair in mammalian cells

In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.     

Long-Tract Mitotic Gene Conversion in Yeast: Evidence for a Triparental Contribution during Spontaneous Recombination

In Saccharomyces cerevisiae, spontaneous mitotic gene conversion at one site is statistically correlated with recombination at other loci. In general, coincident conversion frequencies are highest for tightly linked markers and decline as a function of intermarker distance. Paradoxically, a significant fraction of mitotic gene convertants exhibits concomitant nonreciprocal segregation for multiple and widely spaced markers. We have undertaken a detailed genetic analysis of this class of mitotic recombinants. Our results indicate that mitotic gene conversion in yeast is frequently associated with nonreciprocal segregation of markers centromere-distal to the selected site of conversion. In addition, distal markers are often found to be mosaic within the product colonies. These observations, and others described here, suggest that a percentage of gene conversion in vegetative yeast cells is coupled to a chromosome break and repair mechanism. This hypothesis was further tested using a strain trisomic for chromosome VII which was specially marked to detect homolog-dependent repair events. An association between mitotic gene conversion events and the production of broken chromosomes which are repaired by a homologous-pairing-copy mechanism was supported.     

Recombination and Gene Flux Caused by Gene Conversion and Crossing over in Inversion Heterokaryotypes

A theoretical analysis of the effects of inversions on recombination and gene flux between arrangements caused by gene conversion and crossing over was carried out. Two different mathematical models of recombination were used: the Poisson model (without interference) and the Counting model (with interference). The main results are as follows. (1) Recombination and gene flux are highly site-dependent both inside and outside the inverted regions. (2) Crossing over overwhelms gene conversion as a cause of gene flux in large inversions, while conversion becomes relatively significant in short inversions and in regions around the breakpoints. (3) Under the Counting model the recombination rate between two markers depends strongly on the position of the markers along the inverted segment. Two equally spaced markers in the central part of the inverted segment have less recombination than if they are in a more extreme position. (4) Inversions affect recombination rates in the uninverted regions of the chromosome. Recombination increases in the distal segment and decreases in the proximal segment. These results provide an explanation for a number of observations reported in the literature. Because inversions are ubiquitous in the evolutionary history of many Drosophila species, the effects of inversions on recombination are expected to influence DNA variation patterns.