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Crossing over and chiasma formation during Caenorhabditis elegans meiosis require msh-5, which encodes a conserved germline-specific MutS family member. msh-5 mutant oocytes lack chiasmata between homologous chromosomes, and crossover frequencies are severely reduced in both oocyte and spermatocyte meiosis. Artificially induced DNA breaks do not bypass the requirement for msh-5, suggesting that msh-5 functions after the initiation step of meiotic recombination. msh-5 mutants are apparently competent to repair breaks induced during meiosis, but accomplish repair in a way that does not lead to crossovers between homologs. These results combine with data from budding yeast to establish a conserved role for Msh5 proteins in promoting the crossover outcome of meiotic recombination events. Apart from the crossover deficit, progression through meiotic prophase is largely unperturbed in msh-5 mutants. Homologous chromosomes are fully aligned at the pachytene stage, and germ cells survive to complete meiosis and gametogenesis with high efficiency. Our demonstration that artificially induced breaks generate crossovers and chiasmata using the normal meiotic recombination machinery suggests (1) that association of breaks with a preinitiation complex is not a prerequisite for entering the meiotic recombination pathway and (2) that the decision for a subset of recombination events to become crossovers is made after the initiation step.

The rate of meiotic recombination is not a constant function of physical distance across chromosomes. This variation is manifested by recombination hot spots and cold spots, observed in all organisms ranging from bacteria to humans. It is generally believed that factors such as primary and secondary DNA sequence, as well as chromatin structure and associated proteins, influence the frequency of recombination within a specific region. Several such factors, for example repetitive sequences, gene promoters, or regions with the ability to adopt Z-DNA conformation, have been hypothesized to enhance recombination. However, apart from specific examples, no general trends of association between recombination rates and particular DNA sequence motifs have been reported. In this paper, we analyze the complete sequence data from human chromosome 22 and compare microsatellite repeat distributions with mitotic recombination patterns available from earlier genetic studies. We show significant correlation between long tandem GT repeats, which are known to form Z-DNA and interact with several components of the recombination machinery, and recombination hot spots on human chromosome 22.

A polarized chromosomal arrangement with clustered telomeres in a meiotic prophase nucleus is often called bouquet and is thought to be important for the pairing of homologous chromosomes. Fluorescence in situ hybridization in fission yeast indicated that chromosomal loci are positioned in an ordered manner as anticipated from the bouquet arrangement. Blocking the formation of the telomere cluster with the kms1 mutation created a disorganized chromosomal arrangement, not only for the regions proximal to the telomere but also for interstitial regions. The kms1 mutation also affected the positioning of a linear minichromosome. Consistent with this cytological observation, the frequency of ectopic homologous recombination between a linear minichromosome and a normal chromosome increased in the kms1 background. Intragenic recombination between allelic loci is reduced in the kms1 mutant, but those between non-allelic loci are unaffected or slightly increased. Thus, telomere-led chromosome organization facilitates homologous pairing and also restricts irregular chromosome pairing during meiosis.

Meiotic ectopic recombination occurs at similar frequencies among many sites in the yeast genome, suggesting that all loci are similarly accessible to homology searching. In contrast, we found that his3 sequences integrated in the RDN1 (rDNA) locus were unusually poor participants in meiotic recombination with his3 sequences at other sites. We show that the low rate of meiotic ectopic recombination resulted from the poor ability of RDN1::his3 to act as a donor sequence. SIR2 partially repressed interchromosomal meiotic ectopic recombination at RDN1, consistent with its role in regulating recombination, gene expression, and retrotransposition within RDN1. We propose that RDN1 is physically sequestered from meiotic homology searching mechanisms.

The chromosomal speciation model invokes chromosomal rearrangements as the primary cause of reproductive isolation1. In a heterozygous carrier, chromosomes bearing reciprocal translocations mis-segregate at meiosis, resulting in reduced fertility or complete sterility. Thus, chromosomal rearrangements act as a post-zygotic isolating mechanism. Reproductive isolation in yeast is due to post-zygotic barriers, as many species mate successfully but the hybrids are sterile2,3. Reciprocal translocations are thought to be the main form of large-scale rearrangement since the hypothesized duplication of the whole yeast genome 108 years ago4,5. To test the chromosomal speciation model in yeast, we have characterized chromosomal translocations among the genomes of six closely related species in the Saccharomyces ‘sensu stricto’ complex6. Here we show that rearrangements have occurred between closely related species, whereas more distant ones have colinear genomes. Thus, chromosomal rearrangements are not a prerequisite for speciation in yeast and the rate of formation of translocations is not constant. These rearrangements appear to result from ectopic recombination between Ty elements or other repeated sequences.

The diploid nature of human immunodeficiency virus type 1 (HIV-1) indicates that recombination serves a central function in virus replication and evolution. Previously, while examining the nature of obligatory primer strand transfers during reverse transcription, a high rate of recombination was observed at the ends of the viral genome within the viral long terminal repeats, prompting the following question: does recombination occur at a high rate throughout the genome? To address this question, two vectors based upon different strains of HIV-1 were utilized. The vectors were comprised predominantly of autologous HIV-1 sequence and were approximately the same size as the parental genome. The proviral progeny of heterodimeric virions were analyzed after a single cycle of replication, and the sequence heterogeneity between the two strains allowed direct examination of recombination crossovers. The results obtained indicate that HIV-1 undergoes approximately two to three recombination events per genome per replication cycle. These results imply that both HIV-1 RNAs are typically utilized during reverse transcription and that recombination is an important aspect of HIV-1 replication.

Physical mapping of wheat chromosomes has revealed small chromosome segments of high gene density and frequent recombination interspersed with relatively large regions of low gene density and infrequent recombination. We constructed a detailed genetic and physical map of one highly recombinant region on the long arm of chromosome 5B. This distally located region accounts for 4% of the physical size of the long arm and at least 30% of the recombination along the entire chromosome. Multiple crossovers occurred within this region, and the degree of recombination is at least 11-fold greater than the genomic average. Characteristics of the region such as gene order and frequency of recombination appear to be conserved throughout the evolution of the Triticeae. The region is more prone to chromosome breakage by gametocidal gene action than gene-poor regions, and evidence for genomic instability was implied by loss of gene collinearity for six loci among the homeologous regions. These data suggest that a unique level of chromatin organization exists within gene-rich recombination hot spots. The many agronomically important genes in this region should be accessible by positional cloning.

Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1. Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination. We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects. All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant. Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay. rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers. Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type. Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling. These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination.

Immunoglobulin switch recombination is a specialized recombination event that occurs exclusively in B lymphocytes and is focused on tandemly repetitive DNA sequences called switch regions. Switch recombination occurs as an intrachromosomal deletion event in which the deleted genetic material is excised as a circle. Although the developmental profile of this recombination event is well characterized, the underlying mechanism for switch recombination is poorly understood. Recent studies detected the presence of double strand breaks in switch DNA and the dependency of switching on the Ku and DNA-dependent protein kinase proteins which are involved in repair of double strand breaks by nonhomologous end-joining. Taken together these findings strongly suggest that switch recombination is a specialized recombination system that occurs through a DNA end-joining mechanism.

Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences (gene conversion). This event is often accompanied by a reciprocal exchange between the homologous molecules, resulting in crossing over. The repair of DNA damage by homologous recombination with repeated sequences dispersed throughout the genome might result in chromosomal aberrations or in the inactivation of genes. It is therefore important to understand how the suitable homologous partner for recombination is chosen. We have developed a system in the yeast Saccharomyces cerevisiae that can monitor the fate of a chromosomal double-strand break without the need to select for recombinants. The broken chromosome is efficiently repaired by recombination with one of two potential partners located elsewhere in the genome. One of the partners has homology to the broken ends of the chromosome, whereas the other is homologous to sequences distant from the break. Surprisingly, a large proportion of the repair is carried out by recombination involving the sequences distant from the broken ends. This repair is very efficient, despite the fact that it requires the processing of a large chromosomal region flanking the break. Our results imply that the homology search involves extensive regions of the broken chromosome and is not carried out exclusively by sequences adjacent to the double-strand break. We show that the mechanism that governs the choice of homologous partners is affected by the length and sequence divergence of the interacting partners, as well as by mutations in the mismatch repair genes. We present a model to explain how the suitable homologous partner is chosen during recombinational repair. The model provides a mechanism that may guard the integrity of the genome by preventing recombination between dispersed repeated sequences.

8 pages, 6 figures, 2 tables.-- PMID: 10103269 [PubMed].-- PMCID: PMC91239, We isolated Saccharomyces cerevisiae yeast strains that are able to carry out the second fermentation of sparkling wine from spontaneously fermenting musts in El Penedès (Spain) by specifically designed selection protocols. All of them (26 strains) showed one of two very similar mitochondrial DNA (mtDNA) restriction patterns, whereas their karyotypes differed. These strains showed high rates of karyotype instability, which were dependent on both the medium and the strain, during vegetative growth. In all cases, the mtDNA restriction pattern was conserved in strains kept under the same conditions. Analysis of different repetitive sequences in their genomes suggested that ribosomal DNA repeats play an important role in the changes in size observed in chromosome XII, whereas SUC genes or Ty elements did not show amplification or transposition processes that could be related to rearrangements of the chromosomes showing these sequences. Karyotype changes also occurred in monosporidic diploid derivatives. We propose that these changes originated mainly from ectopic recombination between repeated sequences interspersed in the genome. None of the rearranged karyotypes provided a selective advantage strong enough to allow the strains to displace the parental strains. The nature and frequency of these changes suggest that they may play an important role in the establishment and maintenance of the genetic diversity observed in S. cerevisiae wild populations., This work has been supported by grants from the Spanish Ministry of Education and Science (PB92-0051, PB95-0433, and 95-0012-OP), by additional support from the Generalitat de Catalunya (GRQ93- 8024), and by the Alexander von Humboldt Stiftung (Germany) to B.P. D.N. has been partially supported by fellowships (RE93-05 and RI94-20) from the Generalitat de Catalunya. The firm Ramón Nadal Giró also acknowledges a grant from the Generalitat de Catalunya (IT94/214).

Genetic recombination is the creation of new gene combinations in a cell or gamete, which differ from those of progenitor cells or parental gametes. In eukaryotes, recombination may occur at mitosis or meiosis. Mitotic recombination plays an indispensable role in DNA repair, which presumably directed its early evolution; the multiplicity of recombination genes and pathways may be best understood in this context, although they have acquired important additional functions in generating diversity, both somatically (increasing the immune repertoire) and in germ line (facilitating evolution). Chromosomal homologous recombination and HsRad51 recombinase expression are increased in both immortal and preimmortal transformed cells, and may favor the occurrence of multiple oncogenic mutations. Tumorigenesis in vivo is frequently associated with karyotypic instability, locus-specific gene rearrangements, and loss of heterozygosity at tumor suppressor loci — all of which can be recombinationally mediated. Genetic defects which increase the rate of somatic mutation (several of which feature elevated recombination) are associated with early incidence and high risk for a variety of cancers. Moreover, carcinogenic agents appear to quite consistently stimulate homologous recombination. If cells with high recombination arise, either spontaneously or in response to “recombinogens,” and predispose to the development of cancer, what selective advantage could favor these cells prior to the occurrence of growth-promoting mutations? We propose that the augmentation of telomere-telomere recombination may provide just such an advantage, to hyper-recombinant cells within a population of telomerase-negative cells nearing their replicative (Hayflick) limit, by extending telomeres in some progeny cells and thus allowing their continued proliferation.

Cytogenetic analysis, fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) were applied to characterize the Y-chromosomal breakpoints of three XX male patients. Two of these patients show a breakpoint within a protein kinase gene, PRKY, previously described as a hotspot of ectopic recombination between homologous regions on X and Y chromosomes during male meiosis. The slightly different clinical phenotypes of the three patients cannot be correlated with the localization of the breakpoints.

Centromeres are regions of chromosomes that direct formation of the kinetochore and its subsequent attachment to the spindle, enabling the faithful segregation of the genetic material during cell division. This chromosomal domain, found in all eukaryotes, is functionally conserved but structurally quite divergent between organisms. The overall size and sequence complexity of centromeres generally appears to parallel the developmental complexity of the organism. In Saccharomyces cerevisiae, for example, the centromere is small, consisting of only approximately 200 bp for full function. In higher eukaryotes, however, centromeric regions of the genome not only are much larger (up to 5 Mb in length [see reference 55 for review]) but show no apparent sequence conservation and have been referred to as “regional” centromeres. Despite recent advances in the development of higher eukaryotic experimental systems, relatively little is known about the sequence constituents of centromeres and centric heterochromatin in complex organisms. For Drosophila, the centromere of a minichromosome has been mapped by deletion analysis, and the minimal sequences required for function are now being identified (37, 67). The sequence components necessary for full centromere function appear to include transposable elements of several types as well as low-complexity satellite DNAs (67). The apparent absence of a defined sequence that is responsible for kinetochore formation, as is seen in S. cerevisiae, suggests a redundant, nonsequence-specific initiation event leading to centromere and/or kinetochore formation. There is also evidence in vertebrates for similar redundancy, i.e., Indian muntjac centromeres can be fractionated into multiple individual kinetochore-like units (76). Indirect evidence from humans suggests that megabase arrays of α-satellite or alphoid DNAs, which are a family of A+T-rich 171-bp tandem repeats, are associated with active centromeres (46) and may be sufficient for centromere function (23, 68). Centromere activity can be observed, however, in activated human neocentromeres lacking alphoid repeats (19). The large size of regional centromeres may be important for the additional functions that have been attributed to centromeres of higher eukaryotes, including chromosome adhesion in achiasmate disjunction (32), as well as providing domains of specialized chromatin structure (heterochromatin) in which euchromatic gene transcription and recombination are both repressed. The characteristics of regional centromeric DNA may also reflect an underlying mechanism by which chromatin structure nucleates kinetochore formation. Several regional centromeres display an epigenetic control phenomenon called centromere activation. In the fission yeast Schizosaccharomyces pombe, formation of the centromere into an active state can require multiple cell divisions after introduction of naked minichromosome DNA (66). In other organisms, aberrant chromosomes can form neocentromeres in locations differing from the original centromere, resulting in a mixture of cells containing a chromosome with one site or the other acting as the centromere (1, 6, 69). The regional heterochromatic character of centromeres in complex organisms, however, may result from the accumulation of repeated sequence elements. One consequence of recombinational repression in heterochromatic regions may be the accumulation of mobile genetic elements (13). The prevailing model explaining the observed excess of transposons in centric heterochromatin holds that chromosome rearrangements due to ectopic recombination between similar transposons at different sites in the genome may lead to decreased fitness and selection against such ectopic insertions (12). Alternatively, the insertion of transposons into genic regions may have negative effects on the fitness of individuals in the population, resulting in fewer elements in euchromatic regions of the genome (28). It is possible, however, that repeated sequence elements in some circumstances may have positive effects on fitness rather than just neutral or negative effects. Like centromeric domains, telomeric and subtelomeric regions of most organisms are also regions of specialized chromatin structure and are home to numerous repeated elements (58, 70, 75). The telomerase-elongated simple DNA sequence repeats at the end of the chromosomes of most eukaryotes are essential elements for continued chromosomal end replication and for segregation of the genome. Remarkably, telomeric sequences can function in place of centromeric heterochromatin in Drosophila in the formation of neocentromeres (2, 6, 56, 73). In Drosophila, telomere functions are probably served by the HeT-A and Tart retrotransposons that are found at all chromosomal termini (14, 43). The cell apparently has taken advantage of such elements to serve an essential function. In multicellular fungi, little is known about centromere structure. The chromosomes of the filamentous fungus Neurospora crassa show regions of heavy, intense staining (heterochromatin) in meiosis (44, 64). Centola and Carbon (10) cloned and partially characterized a contiguous set of artificial yeast chromosomes (YACs) containing DNA that spanned the centromere region of linkage group (LG) VII of Neurospora. This region, approximately 450 kb in length, was found to be both A+T rich and recombination deficient. In addition, they identified a centromere-specific repeated DNA sequence. Comparison of the sequence of this centromere-specific clone to homologous DNAs from elsewhere in the genome suggested that they had undergone repeat-induced point mutation (RIP), a process that scans the genome of Neurospora for repeated DNAs during the sexual cycle and induces GC to AT transition mutations, and often DNA methylation, specific to the duplicated sequences (61). In this study, we have further characterized the centromere region of LG VII of Neurospora and have discovered a nested cluster of putative transposable elements and simple sequence repeats. A repeated DNA sequence previously found to map to centromere-linked regions of the Neurospora genome (10) is now shown to be a copia-like element (named Tcen) which is novel in that it is the only known transposon to be shown to map exclusively to centromere regions. In addition, the region contains the degenerate remains of several other transposons, as well as three different low-complexity DNAs organized in a tightly nested arrangement. Although these features have yet to be associated with kinetochore formation, the structural similarity of the Neurospora centromere VII region to the centromere of the Drosophila Dp1187 minichromosome (37, 67) suggests that Neurospora kinetochore-forming regions may be similarly redundant and nonspecific.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.

Several features indicate that the low polymorphic human minisatellite MsH42 region could be involved in recombination. It contains different well-known recombination motifs, is able to generate single-stranded loops and is specifically recognized by nuclear proteins. These characteristics led us to investigate the possible recombinogenic activity of the MsH42 region in terms of intramolecular recombination. We constructed two plasmids, one of them carrying two copies of the minisatellite region and the other one containing sequences upstream of this repetitive region. We showed that MsH42 strongly stimulates intramolecular in vitro recombination, approximately 22 times more than the control sequence, solely when the source of biological extract is mouse testes, suggesting that MsH42 could be a hotspot involved in meiotic recombination. Furthermore, there is a direct relationship between the frequency of equal crossovers and the enhancement of recombination. Interestingly, the third repeat of the minisatellite array is always involved in the resolution of unequal crossovers leading to minisatellite shortening. As far as we know, our results provide the first evidence that a non-hypervariable minisatellite can enhance homologous recombination.

Duplicated genes and repetitive sequences are distributed throughout the genomes of complex organisms. The homology between related sequences can promote nonallelic (ectopic) recombination, including gene conversion and reciprocal exchange. Resolution of these events can result in translocations, deletions, or other harmful rearrangements. In yeast, ectopic recombination between sequences on nonhomologous chromosomes occurs at high frequency. Because the mammalian genome is replete with duplicated sequences and repetitive elements, high levels of ectopic exchange would cause aneuploidy and genome instability. To understand the factors regulating ectopic recombination in mice, we evaluated the effects of homology length on gene conversion between unlinked sequences in the male germline. Previously, we found high levels of gene conversion between lacZ transgenes containing 2557 bp of homology. We report here that genetic background can play a major role in ectopic recombination; frequency of gene conversion was reduced by more than an order of magnitude by transferring the transgenes from a CF1 strain background to C57BL/6J. Additionally, conversion rates decreased as the homology length decreased. Sequences sharing 1214 bp of sequence identity underwent ectopic conversion less frequently than a pair sharing 2557 bp of identity, while 624 bp was insufficient to catalyze gene conversion at significant levels. These results suggest that the germline recombination machinery in mammals has evolved in a way that prevents high levels of ectopic recombination between smaller classes of repetitive sequences, such as the Alu family. Additionally, genomic location appeared to influence the availability of sequences for ectopic recombination.

Background: Rad51 and Dmc1 are Saccharomyces cerevisiae homologues of the Escherichia coli recombination protein RecA. Mutant analysis has shown that both proteins are required for normal meiotic recombination, for timely and efficient formation of synaptonemal complex and for normal progression out from meiotic prophase. Results: We have further characterized rad51 and dmc1 single mutants. A dmc1 mutation confers more severe defects in double strand break (DSB) resolution, crossover recombination and meiotic progression than does a rad51 mutant; in contrast, during return to growth, a rad51 mutation confers more severe defects in viability and intrachromosomal recombination than does a dmc1 mutation. Analysis of a rad51 dmc1 double mutant, in parallel with single mutants, shows that the double mutant is more defective with respect to the formation of crossovers during meiosis and, especially strikingly, with respect to interhomologue and intrachromosomal recombination during return to growth. Consistent with the observation of DMC1-dependent recombination in a rad51 mutant, subnuclear complexes of Dmc1 protein were detected for the first time in this mutant. In contrast to the effects on recombination, the effect of the double mutant on meiotic progression was similar to that of the rad51 single mutant. Conclusion: Rad51 and Dmc1 each make unique contributions to meiotic recombination. However, the two proteins are capable of substituting for one another under some circumstances, implying that they most likely share at least one recombination function. Recombination and cell cycle phenotypes are all consistent with the possibility that a dmc1 mutation causes an arrest of the post-DSB recombination complexes at a later, more stable stage than does a rad51 mutation.

Homologous recombination occurs at high frequency during meiosis and is essential for the proper segregation of chromosomes and the generation of genetic diversity. Meiotic recombination is controlled in numerous ways. In the fission yeast Schizosaccharomyces pombe nutritional starvation induces meiosis and high-level expression of many genes, including numerous recombination (rec) genes, whose products are required for recombination. Accompanying the two meiotic divisions are profound changes in nuclear and chromosomal structure and movement, which may play an important role in meiotic recombination. Although recombination occurs throughout the genome, it occurs at high frequency in some intervals (hotspots) and at low frequency in others (coldspots). The well-characterized hotspot M26 is activated by the Mts1/Mts2 protein; this site and its binding proteins interact with the local chromosomal structure to enhance recombination. A coldspot between the silent mating-type loci is repressed by identified proteins, which may also alter local chromatin. We discuss in detail the rec genes and the possible functions of their products, some but not all of which share homology with other identified proteins. Although some of the rec gene products are required for recombination throughout the genome, others demonstrate regional specificity and are required in certain genomic regions but not in others. Throughout the review contrasts are made with meiotic recombination in the more thoroughly studied budding yeast Saccharomyces cerevisiae.

P-element-induced recombination in Drosophila melanogaster occurs premeiotically. Recombinants are therefore expected to accumulate in the stem cells of the germline of Pelement-carrying males. We show that both the recombination frequency and the incidence of “clustering” increase with the age of males carrying various P-element derivatives. The combination of end-deleted elements can lead to average recombination frequencies >50% with individual instances of 100% recombination. These elements also lowered the fertility of the carriers. We investigated these features by constructing an analytical and a computer simulation model of the course of events in the germline, incorporating the recently proposed hybrid element insertion (HEI) model of Pelement activity. The model is able to predict extreme recombination levels, segregation ratio biases and lowered fertility through cell death in a single analysis.

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

The bronze (bz) gene is a recombinational hotspot in the maize genome: its level of meiotic recombination per unit of physical length is > 100-fold higher than the genome9s average and is the highest of any plant gene analyzed to date. Here, we examine whether recombination is also unevenly distributed within the bz gene. In yeast genes, recombination (conversion) is polarized, being higher at the end of the gene where recombination is presumably initiated. We have analyzed products of meiotic recombination between heteroallelic pairs of bz mutations in both the presence and absence of heterologies and have sequenced the recombination junction in 130 such Bz intragenic recombinants. We have found that in the absence of heterologies, recombination is proportional to physical distance across the bz gene. The simplest interpretation for this lack of polarity is that recombination is initiated randomly within the gene. Insertion mutations affect the frequency and distribution of intragenic recombination events at bz, creating hotspots and coldspots. Single base pair heterologies also affect recombination, with fewer recombination events than expected by chance occurring in regions of the bz gene with a high density of heterologies. We also provide evidence that meiotic recombination in maize is conservative, that is, it does not introduce changes, and that meiotic conversion tracts are continuous and similar in size to those in yeast.

One of the oldest hypotheses for the advantage of recombination is that recombination allows beneficial mutations that arise in different individuals to be placed together on the same chromosome. Unless recombination occurs, one of the beneficial alleles is doomed to extinction, slowing the rate at which adaptive mutations are incorporated within a population. We model the effects of a modifier of recombination on the fixation probability of beneficial mutations when beneficial alleles are segregating at other loci. We find that modifier alleles that increase recombination do increase the fixation probability of beneficial mutants and subsequently hitchhike along as the mutants rise in frequency. The strength of selection favoring a modifier that increases recombination is proportional to lambda(2)S delta r/r when linkage is tight and lambda(2)S3 delta r/N when linkage is loose, where lambda is the beneficial mutation rate per genome per generation throughout a population of size N, S is the average mutant effect, r is the average recombination rate, and delta r is the amount that recombination is modified. We conclude that selection for recombination will be substantial only if there is tight linkage within the genome or if many loci are subject to directional selection as during periods of rapid evolutionary change.

Illegitimate (nonhomologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. Under special conditions in Escherichia coli, we have found a new type of illegitimate recombination that requires an interaction between homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type II restriction in vitro and type I (EcoKI) restriction in vivo within a delta rac recBC recG ruvC strain. Removal of one of the repeats or its replacement with heterologous DNA resulted in a reduction in the level of recombination. The recombining sites themselves shared, at most, a few base pairs of homology. Many of the recombination events joined a site in one of the repeats with a site in another repeat. In two of the products, one of the recombining sites was at the end of one of the repeats. Removal of one of the EcoKI sites resulted in decreased recombination. We discuss the possibility that some structure made by homologous interaction between the long repeats is used by the EcoKI restriction enzyme to promote illegitimate recombination. The possible roles and consequences of this type of homologous interaction are discussed.

Non-recombining populations should suffer from four classic population genetic disadvantages: (1) they cannot reverse Muller's Ratchet, the accumulation of deleterious mutations caused by genetic drift and mutation; (2) whenever the fix a favourable mutation they lose all unlinked favourable variants; (3) they tend to lose favourable mutations that are linked to deleterious mutations; and (4) their genetic loads can be quite high when deleterious mutations have synergistic effects. It is commonly assumed that inter-chromosomal recombination (independent assortment) can counter these phenomena, but this has been studied only for the genetic load case. In contrast, many studies have shown that recombination via crossing over can counter these phenomena. Here we first show that segregation alone can strongly decelerate Muller's Ratchet in diploids, i.e. that recombination is not the only way to do so. We then show that inter-chromosomal recombination can indeed deal with phenomena (1) to (3) above very effectively if the genome consists of a moderate number of chromosomes. Therefore, if the above advantages of genetic recombination played a large role in the initial success of eukaryotic sex, the crucial moment in the origin of sex might have been the evolution of inter-chromosomal recombination, i.e. the evolution of genome segmentation, segregation, and syngamy. Crossing over might have become established as a major recombinational device only later, eliminating the disadvantages of extensively segmented genomes.

Studies of meiotic recombination have revealed an evolutionary paradox. Molecular and genetic analysis has shown that crossing over initiates at specific sites called hotspots, by a recombinational-repair mechanism in which the initiating hotspot is replaced by a copy of its homolog. We have used computer simulations of large populations to show that this mechanism causes active hotspot alleles to be rapidly replaced by inactive alleles, which arise by rare mutation and increase by recombination-associated conversion. Additional simulations solidified the paradox by showing that the known benefits of recombination appear inadequate to maintain its mechanism. Neither the benefits of accurate segregation nor those of recombining flanking genes were sufficient to preserve active alleles in the face of conversion. A partial resolution to this paradox was obtained by introducing into the model an additional, nonmeiotic function for the sites that initiate recombination, consistent with the observed association of hotspots with functional sites in chromatin. Provided selection for this function was sufficiently strong, active hotspots were able to persist in spite of frequent conversion to inactive alleles. However, this explanation is unsatisfactory for two reasons. First, it is unlikely to apply to obligately sexual species, because observed crossover frequencies imply maintenance of many hotspots per genome, and the viability selection needed to preserve these would drive the species to extinction. Second, it fails to explain why such a genetically costly mechanism of recombination has been maintained over evolutionary time. Thus the paradox persists and is likely to be resolved only by significant changes to the commonly accepted mechanism of crossing over.

Homologous recombination pathways probably evolved primarily to accomplish chromosomal repair and the formation and resolution of duplications by sister-chromosome exchanges. Various DNA lesions initiate these events. Classical recombination assays, involving bacterial sex, focus attention on double-strand ends of DNA. Sexual exchanges, initiated at these ends, depend on the RecBCD pathway. In the absence of RecBCD function, mutation of the sbcB and sbcC genes activates the apparently cryptic RecF pathway. To provide a more general view of recombination, we describe an assay in which endogenous DNA damage initiates recombination between chromosomal direct repeats. The repeats flank markers conferring lactose utilization (Lac+) and ampicillin resistance (ApR); recombination generates Lac-ApS segregants. In this assay, the RecF pathway is not cryptic; it plays a major role without sbcBC mutations. Others have proposed that single-strand gaps are the natural substrate for RecF-dependent recombination. Supporting this view, recombination stimulated by a double-strand break (DSB) in a chromosomal repeat depended on RecB function, not RecF function. Without RecBCD function, sbcBC mutations modified the RecF pathway and allowed it to catalyze DSB-stimulated recombination. Sexual recombination assays overestimate the importance of RecBCD and DSBs, and underestimate the importance of the RecF pathway.

Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

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.

We have shown elsewhere that there is no, or very little, homology at the recombination sites in DNA gyrase-mediated illegitimate recombination in vitro. On the other hand, many reports have indicated that illegitimate recombination takes place between sequences with a short homology. To clarify this contradiction, we analyzed the mechanism of DNA gyrase-mediated illegitimate recombination in vivo, by isolating a temperature-sensitive gyrA mutant (gyrAhr1) that causes spontaneous illegitimate recombination at a higher frequency than that of the wild-type. This mutant also causes spontaneous induction of lambda prophage. It is therefore suggested that the gyrAhr1 mutation induces strand breaks in the chromosome, resulting in the formation of illegitimate recombinants. Analysis of the recombination junctions of lambdabio transducing phages formed spontaneously in the gyrAhr1 mutant revealed that the Escherichia coli bio and lambda recombination sites have an average homologous sequence of only 1.3 base pairs. This is the first indication that homology in vivo is not required for illegitimate recombination. On the other hand, a short homology of 8.4 bp, on average, was found in the junctions of lambdabio transducing phages formed spontaneously in the wild-type bacteria. When the gyrAhr1 mutant was irradiated with UV, short homologies were also detected in the junctions. We concluded that illegitimate recombination, which takes place spontaneously in the gyrAhr1 mutants, is distinguishable from spontaneous recombination in the wild-type and from UV-induced recombination in the mutant with regard to the requirement for short homology. We propose that short-homology-independent illegitimate recombination is mediated by subunit exchange between DNA gyrase, while short-homology-dependent recombination is triggered by double-strand breaks and completed by processing, annealing, and ligation of DNA ends.

Replication, DNA organization, and mismatch repair (MMR) can influence recombination. We examined the effects of altered replication due to a mutation in the polymerase delta gene, long inverted repeats (LIRs) in motifs similar to those in higher eukaryotes, and MMR on intrachromosomal recombination between highly diverged (28%) truncated genes in Saccharomyces cerevisiae. A combination of altered replication and an LIR increased recombination up to 700-fold, while each alone led to a 3- to 20-fold increase. Homeologous recombination was not altered by pms1, msh2, and msh3 mismatch repair mutations. Similar to our previous observations for replication slippage-mediated deletions, there were > or = 5-bp identical runs at the recombination breakpoints. We propose that the dramatic increase in recombination results from enhancement of the effects of altered replication by the LIR, leading to recombinationally active initiating structures. Such interactions predict replication-related, MMR-independent genome changes.

Meiotic homologous recombination serves three principal roles. First, recombination reas-sorts the linkages between newly-arising alleles to provide genetic diversity upon which natural selection can act. Second, recombination is used to repair certain types of DNA damage to provide a mechanism of genomic homeostasis. Third, with few exceptions homologous recombination is required for the appropriate segregation of homologous chromosomes during meiosis. Recombination rates are elevated near DNA sites called “recombination hotspots.” These sites influence the distribution of recombination along chromosomes and the timing of recombination during the life cycle. Recent advances have revealed biochemical steps of hotspot activation and have suggested that hotspots may regulate when and where recombination occurs. Two models for hotspot activation, one in which hotspots act early in the recombination pathway and one in which hotspots act late in the recombination pathway, are presented. The latter model can account for changes at hypervariable minisatellite DNA in metazoan genomes by invoking resolution of Holliday junctions at mini-satellite DNA repeats.

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.

Recombination during meiosis in the form of crossover events promotes the segregation of homologous chromosomes by providing the only physical linkage between these chromosomes. Recombination occurs not only between allelic sites but also between non-allelic (ectopic) sites. Ectopic recombination is often suppressed to prevent non-productive linkages. In this study, we examined the effects of various mutations in genes involved in meiotic recombination on ectopic recombination during meiosis. RAD24, a DNA damage checkpoint clamp-loader gene, suppressed ectopic recombination in wild type in the same pathway as RAD51. In the absence of RAD24, a meiosis-specific recA homolog, DMC1, suppressed the recombination. Homology search and strand exchange in ectopic recombination occurred when either the RAD51 or the DMC1 recA homolog was absent, but was promoted by RAD52. Unexpectedly, the zip1 mutant, which is defective in chromosome synapsis, showed a decrease, rather than an increase, in ectopic recombination. Our results provide evidence for two types of ectopic recombination: one that occurs in wild-type cells and a second that occurs predominantly when the checkpoint pathway is inactivated.

Although homologous recombination is an important pathway for the repair of double-stranded DNA breaks in mitotically dividing eukaryotic cells, these events can also have negative consequences, such as loss of heterozygosity (LOH) of deleterious mutations. We mapped about 140 spontaneous reciprocal crossovers on the right arm of the yeast chromosome IV using single-nucleotide-polymorphism (SNP) microarrays. Our mapping and subsequent experiments demonstrate that inverted repeats of Ty retrotransposable elements are mitotic recombination hotspots. We found that the mitotic recombination maps on the two homologs were substantially different and were unrelated to meiotic recombination maps. Additionally, about 70% of the DNA lesions that result in LOH are likely generated during G1 of the cell cycle and repaired during S or G2. We also show that different genetic elements are associated with reciprocal crossover conversion tracts depending on the cell cycle timing of the initiating DSB., Author Summary Double-strand breaks (DSBs) are DNA lesions that can be fatal to a cell if left unrepaired. They can be caused by exogenous sources, such as gamma radiation, or endogenous stresses, such as high levels of transcription. Yeast cells primarily repair DSBs that are initiated outside of meiosis by mitotic recombination, which can result in physical exchanges between chromosomes, known as crossovers. We created a mitotic recombination map of one chromosome arm, representing 10% of the genome. This recombination map allows us to determine which regions of the chromosome arm are more susceptible to DNA damage than other regions. We were able to determine that most DSBs that result in detectable genomic changes were initiated prior to DNA replication and that some secondary DNA structures can be recombination hotspots. Recombination can also occur during meiosis, as a method of ensuring proper chromosome segregation. However, previously reported meiotic recombination maps have no correlation with our mitotic recombination map.

In the yeast Saccharomyces cerevisiae ectopic recombination has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family. Little is known about the mechanism(s) that prevent recombination between repeated sequences. We have previously shown that nonreciprocal recombination (gene conversion) of a genetically marked Ty can be induced either by the presence of high levels of Ty cDNA or by transcription of the marked Ty from a GAL1 promoter. These two kinds of induction act in a synergistic manner. To further characterize these two kinds of Ty recombination, we have investigated the role played by the RkD52 and RAD1 genes. We have found that the RAD52 and RAD1 gene products are essential to carry out transcription-induced Ty conversion whereas cDNA-mediated conversion can take place in their absence.

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.

Zip1 is a yeast synaptonemal complex (SC) central region component and is required for normal meiotic recombination and crossover interference. Physical analysis of meiotic recombination in a zip1 mutant reveals the following: Crossovers appear later than normal and at a reduced level. Noncrossover recombinants, in contrast, seem to appear in two phases: (i) a normal number appear with normal timing and (ii) then additional products appear late, at the same time as crossovers. Also, Holliday junctions are present at unusually late times, presumably as precursors to late-appearing products. Red1 is an axial structure component required for formation of cytologically discernible axial elements and SC and maximal levels of recombination. In a red1 mutant, crossovers and noncrossovers occur at coordinately reduced levels but with normal timing. If Zip1 affected recombination exclusively via SC polymerization, a zip1 mutation should confer no recombination defect in a red1 strain background. But a red1 zip1 double mutant exhibits the sum of the two single mutant phenotypes, including the specific deficit of crossovers seen in a zip1 strain. We infer that Zip1 plays at least one role in recombination that does not involve SC polymerization along the chromosomes. Perhaps some Zip1 molecules act first in or around the sites of recombinational interactions to influence the recombination process and thence nucleate SC formation. We propose that a Zip1-dependent, pre-SC transition early in the recombination reaction is an essential component of meiotic crossover control. A molecular basis for crossover/noncrossover differentiation is also suggested.

Junctions at class switch recombination sites in the genome are characterized by a unique sequence feature. Nucleotide substitutions and small deletions are common on either of the two sides of the switch junction, but not on both together. We have previously reported an extrachromosomal substrate assay system for analyzing the recombination of class switch sequences. Here we have sequenced nine junctions on each side of the break point and compared these to 17 recombination junctions of control substrates from the same cells. Five of the nine switch recombination junctions have nucleotide substitutions and deletions, with multiple nucleotide changes being more common. Furthermore, mutations were found only on a single side of the junction, just as for the recombination of switch sequences in the genome. In contrast, only one of 17 control substrate junctions had a mutation, and this was a single nucleotide insertion. This difference is highly significant (P < 0.00007) and indicates that the fundamental recombination mechanism is likely to be similar for switch sequences in the chromosome and on minichromosome substrates.

The function of meiotic recombination has remained controversial, despite recent inroads into mechanisms. Ideas concerning a possible role of recombination in the elimination or efficient incorporation of mutations have been backed by theoretical studies but have lacked empirical support. Recent investigations into the basis for local variations in recombination frequency in yeast have uncovered a strong association between recombination initiation sites and transcriptional regulatory sequences. Other recent studies indicate a strong correlation between transcription and mutation rates in yeast genes. Taken together, these data imply that distributions of recombination and mutation frequencies may be strongly correlated. This suggests that recombination may be targeted to genomic sites of high mutation frequency; such a ‘mutation-tracking’ function would clearly aid in the shuffling of mutations to break up unfavorable and create favorable allelic combinations. Moreover, recent insights into the mechanism of gene conversion in yeast reveal a very strong inherent bias in favor of alleles on the non-initiating homolog. Combined with mutation tracking, these findings suggest a novel and general mechanism by which allelic gene conversion may act to eliminate mutations.

Publisher Summary This chapter discusses the distinct features of recA -independent recombination among repetitive sequences in bacteria, with special emphasis on replicational models and their implications in related processes such as genome rearrangement and gene amplification in eukaryotic cells. In Escherichia coli , general (or homologous) recombination typically involves extensive stretches of sequence homology and is dependent on the RecA protein and other recombination functions. Site-specific recombination occurs at highly preferred sites and requires special proteins other than RecA and other activities involved in general recombination. Intramolecular reciprocal exchange among direct repeats can lead to the deletion of one of the repeats and any intervening sequence, whereas intermolecular unequal crossover can result in deletion in one of the products and addition in the other. These processes can be accomplished by RecA-mediated general homologous recombination. It has been shown that efficient recA -independent recombination can also occur at direct repeats, resulting in deletion and addition, which are sometimes accompanied by other rearrangement(s). recA -independent recombination is most efficient when the direct repeats are in close proximity and it can form products different from that of recA -dependent homologous recombination.

Recombination is widely considered to linearly depend on the length of the homologous sequences. An 11% mismatch decreases the rate of phage-plasmid recombination 240-fold. Two single nucleotide mismatches, which reduce the longest uninterrupted stretch of similarity from 232 base pairs (bp) to 134 bp, reduce gene conversion in mouse L cells 20-fold. The efficiency of gene targeting through homologous recombination in mouse embryonic stem cells can be increased by using an isogenic, rather than a non-isogenic, DNA construct. In this study we asked whether a high degree of sequence identity between homologous mouse chromosomes enhances meiotic recombination at a hotspot. Sites of meiotic recombination in the mouse major histocompatibility complex (MHC) class II region are not randomly distributed but are almost all clustered within short segments known as recombinational hotspots. The wm7 MHC haplotype, derived from Japanese wild mice Mus musculus molossinus, enhances meiotic recombination at a hotspot near the Lmp2 gene. Heterozygotes between the wm7 haplotype and the b or k haplotypes have yielded a high frequency of recombination (2.1%) in 1.3 kilobase kb segment of this hotspot. 20 refs., 2 figs.

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.

During meiosis, crossovers occur at a high level, but the level of noncrossover recombinants is even higher. The biological rationale for the existence of the latter events is not known. It has been suggested that a noncrossover-specific pathway exists specifically to mediate chromosome pairing. Using a physical assay that monitors both crossovers and noncrossovers in cultures of yeast undergoing synchronous meiosis, we find that both types of products appear at essentially the same time, after chromosomes are fully synapsed at pachytene. We have also analyzed a situation in which commitment to meiotic recombination and formation of the synaptonemal complex are coordinately suppressed (mer1 versus mer1 MER2++). We find that suppression is due primarily to restoration of meiosis-specific double-strand breaks, a characteristic of the major meiotic recombination pathway. Taken together, the observations presented suggest that there probably is no noncrossover-specific pathway and that restoration of intermediate events in a single pairing/recombination pathway promotes synaptonemal complex formation. The biological significant of noncrossover recombination remains to be determined, however.

Homologous recombination occurs intrachromosomally as well as interchromosomally, both in mitotic (somatic) cells as well as meiotically in the germline. These different processes can serve very different purposes in maintaining the integrity of the organism and in enhancing diversity in the species. As shown here, comparison of the frequencies of intra- and interchromosomal recombination in meiotic and mitotic cells of both mouse and yeast argues that interchromosomal recombination is particularly low in mitotic cells of metazoan organisms. This result in turn suggests that the recombination machinery of metazoa might be organized to avoid the deleterious effects of homozygotization in somatic cells while still deriving the benefits of species diversification and of DNA repair.

Two hot spots of ectopic Xp-Yp recombination have previously been shown to be at the origin of XX maleness (Well et al., 1994, Nature Genet. 7:414-419). To get more insight into the molecular basis of the abnormal interchange, 25 Y(+) class 3 XX male patients were studied. The two hot spots were found to account for the aberrant exchange in more than 50% of the cases. In addition, there is a correlation between the prevalence of each hot spot and the degree of X-Y homology between the corresponding fragments. Sequencing of the X-Y junctions in six patiens, who carried a breakpoint mapping in either of these two hot spot fragments, showed that their precise lacations were different from one individual to the other. In particular, the results obtained here in four new patients exclude the possibility that the repeated elements, present in these X-Y homologous fragments, are responsible for the high incidence of X-Y interchanges observed. Moreover, the breakpoints of all 25 class 3 XX males were found to be arranged in the same order on the X and Y chromosomes. This suggests that most ectopic recombinations leading to class 3 XX maleness involve X-Y homologous sequences persisting from an ancestral larger block of homology on the short arms of both sex chromosomes.

Intrachromosomal recombination between direct repeats can occur either as gene conversion events, which maintain exactly the number of repeat units, or as deletions, which reduce the number of repeat units. Gene conversions are classical recombination events that utilize the standard chromosome recombination machinery. Spontaneous deletions between direct repeats are generally recA-independent in E. coli and RAD52-independent in S. cerevisiae. This independence from the major recombination genes does not mean that deletions form through a nonrecombinational process. Deletions have been suggested to result from sister chromatid exchange at the replication fork in a recA-independent process. The same type of exchange is proposed to be RAD52-independent in Saccharomyces cerevisiae. RAD52-dependent events encompass all events that involve the initial steps of a recombination reaction, which include strand invasion to form a heteroduplex intermediate.

Publisher Summary This chapter reviews the origin of numerical chromosome abnormalities. Humans appear to be unique in their very high frequency of numerical chromosome abnormalities. The recent development of large numbers of molecular probes for all human chromosomes has provided the tools to enable the mechanisms underlying the high frequency of aneuploidy to be investigated. Earlier observations have concentrated on a small number of chromosomes, mainly those that are most frequently associated with aneuploidy. These investigations have shown a very wide diversity of mechanisms underlying numerical chromosome abnormalities. Also, the relative importance of the different mechanisms differs widely among chromosomes. With the exception of the 45,X, 47,XXY, and 47,XYY aneuploids, nondisjunction rarely involves a paternal chromosome and for the autosomes the proportion due to nondisjunction of a maternal chromosome ranges from 90 to 100%. The trisomies resulting from an error at maternal meiosis, a number of different mechanisms have been observed. These include errors of maternal MI (mat MI) and mat MII in which the nondisjoined chromosomes/chromatids show normal recombination, and errors of mat MI and mat MII in which the nondisjoined chromosome/chromatids show abnormal recombination. Abnormal recombination includes total absence of recombination, reduced recombination, and abnormally high levels of pericentromeric recombination.

The pairing and recombination of homologous chromosomes is crucial for their reductional segregation during meiosis I, but until now it has been unclear how chromosomes recognize their homologous partners. Ding et al. observed that fluorescently tagged sme2 loci, located on Schizosaccharomyces pombe chromosome 2, paired frequently in early meiotic prophase. When the sme2 locus was translocated to another region, robust pairing was seen at this ectopic site, indicating that sme2 is sufficient to induce robust homologous pairing. Furthermore, when two copies of sme2 were present on non-homologous chromosomes, the two loci associated transiently, suggesting that sme2 loci can recognize each other. Importantly, sme2 non-coding RNA transcripts, transcribed from both homologous chromosomes, accumulated at their respective gene loci and were found to be essential for pairing. Whether other pairing sites also exist on chromosome 2 remains to be determined.