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International audience; Centromeres are critical for cell division, loading CENH3 or CENPA histone variant nucleosomes, directing kinetochore formation and allowing chromosome segregation(1,2). Despite their conserved function, centromere size and structure are diverse across species. To understand this centromere paradox(3,4), it is necessary to know how centromeric diversity is generated and whether it reflects ancient trans-species variation or, instead, rapid post-speciation divergence. To address these questions, we assembled 346 centromeres from 66 Arabidopsis thaliana and 2 Arabidopsis lyrata accessions, which exhibited a remarkable degree of intra- and inter-species diversity. A. thaliana centromere repeat arrays are embedded in linkage blocks, despite ongoing internal satellite turnover, consistent with roles for unidirectional gene conversion or unequal crossover between sister chromatids in sequence diversification. Additionally, centrophilic ATHILA transposons have recently invaded the satellite arrays. To counter ATHILA invasion, chromosome-specific bursts of satellite homogenization generate higher-order repeats and purge transposons, in line with cycles of repeat evolution. Centromeric sequence changes are even more extreme in comparison between A. thaliana and A. lyrata. Together, our findings identify rapid cycles of transposon invasion and purging through satellite homogenization, which drive centromere evolution and ultimately contribute to speciation.

Meiotic recombination is essential for chromosome segregation at meiosis and fertility. It is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homologue of an archaeal topoisomerase (Topo VIA)1. Here we describe previously uncharacterized Spo11-induced lesions, 34 to several hundred base pair-long gaps, which are generated by coordinated pairs of DSBs termed double DSBs. Isolation and genome-wide mapping of the resulting fragments with single base-pair precision revealed enrichment at DSB hotspots but also a widely dispersed distribution across the genome. Spo11 prefers to cut sequences with similarity to a DNA-bending motif2, which indicates that bendability contributes to the choice of cleavage site. Moreover, fragment lengths have a periodicity of approximately (10.4n + 3) base pairs, which indicates that Spo11 favours cleavage on the same face of underwound DNA. Consistently, double DSB signals overlap and correlate with topoisomerase II-binding sites, which points to a role for topological stress and DNA crossings in break formation, and suggests a model for the formation of DSBs and double DSBs in which Spo11 traps two DNA strands. Double DSB gaps, which make up an estimated 20% of all initiation events, can account for full gene conversion events that are independent of both Msh2-dependent heteroduplex repair3,4 and the MutLγ endonuclease4. Because non-homologous gap repair results in deletions, and ectopically re-integrated double DSB fragments result in insertions, the formation of double DSBs is a potential source of evolutionary diversity and pathogenic germline aberrations. Meiotic recombination in yeast is not only initiated by single break sites, but also caused by closely spaced Spo11-dependent double-stranded DNA breaks that create chromosomal gaps.

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity

We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the first metatherian ('marsupial') species to be sequenced, the opossum provides a unique perspective on the organization and evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In contrast, about 20% of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.

The human Y chromosome, transmitted clonally through males, contains far fewer genes than the sexually recombining autosome from which it evolved. The enormity of this evolutionary decline has led to predictions that the Y chromosome will be completely bereft of functional genes within ten million years. Although recent evidence of gene conversion within massive Y-linked palindromes runs counter to this hypothesis, most unique Y-linked genes are not situated in palindromes and have no gene conversion partners. The 'impending demise' hypothesis thus rests on understanding the degree of conservation of these genes. Here we find, by systematically comparing the DNA sequences of unique, Y-linked genes in chimpanzee and human, which diverged about six million years ago, evidence that in the human lineage, all such genes were conserved through purifying selection. In the chimpanzee lineage, by contrast, several genes have sustained inactivating mutations. Gene decay in the chimpanzee lineage might be a consequence of positive selection focused elsewhere on the Y chromosome and driven by sperm competition.

Eight palindromes comprise one-quarter of the euchromatic DNA of the male-specific region of the human Y chromosome, the MSY. They contain many testis-specific genes and typically exhibit 99.97% intra-palindromic (arm-to-arm) sequence identity. This high degree of identity could be interpreted as evidence that the palindromes arose through duplication events that occurred about 100,000 years ago. Using comparative sequencing in great apes, we demonstrate here that at least six of these MSY palindromes predate the divergence of the human and chimpanzee lineages, which occurred about 5 million years ago. The arms of these palindromes must have subsequently engaged in gene conversion, driving the paired arms to evolve in concert. Indeed, analysis of MSY palindrome sequence variation in existing human populations provides evidence of recurrent arm-to-arm gene conversion in our species. We conclude that during recent evolution, an average of approximately 600 nucleotides per newborn male have undergone Y-Y gene conversion, which has had an important role in the evolution of multi-copy testis gene families in the MSY.

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae1. In vertebrate cells, little is known about the exact function of the Mre11–Rad50–Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal2. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants3. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.

Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks. Although BRCA1 and BRCA2 affect replication fork processing, direct evidence that BRCA gene products regulate homologous recombination at stalled chromosomal replication forks is lacking, due to a dearth of tools for studying this process. Here we report that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mouse cells. Tus/Ter-induced homologous recombination entails processing of bidirectionally arrested forks. We find that the Brca1 carboxy (C)-terminal tandem BRCT repeat and regions of Brca1 encoded by exon 11-two Brca1 elements implicated in tumour suppression-control Tus/Ter-induced homologous recombination. Inactivation of either Brca1 or Brca2 increases the absolute frequency of 'long-tract' gene conversions at Tus/Ter-stalled forks, an outcome not observed in response to a site-specific endonuclease-mediated chromosomal double-strand break. Therefore, homologous recombination at stalled forks is regulated differently from homologous recombination at double-strand breaks arising independently of a replication fork. We propose that aberrant long-tract homologous recombination at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells.

Genetic approaches to manipulating or eradicating disease vectors have been proposed as alternatives to malaria eradication. The success of this approach depends on efficient spread of a genetic modification in field populations. Windbichler et al. show that a synthetic genetic element consisting of mosquito regulatory elements and the homing endonuclease gene I-SceI can spread from a small number of individual Anopheles gambiae mosquitoes into large receptive populations in just a few generations. This is the first demonstration of a synthetic gene drive system in the main human malaria vector — and a similar approach should be applicable to many other pest species. Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity1,2,3. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof of principle, the possibility of targeting the mosquito’s ability to serve as a disease vector4,5,6,7. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations8. We have suggested previously that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose9. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions10 and the homing endonuclease gene I-SceI11,12,13, can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to invade receptive mosquito cage populations rapidly, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.

Sleeping sickness is caused by the trypanosome parasite. This parasite outwits the human immune system by periodically changing its coat protein in a process known as variant surface glycoprotein (VSG) switching. The favoured model to explain switching invokes a normal host process, called gene conversion, that is used to repair DNA. In this work, Nina Papavasiliou and colleagues establish the first in vitro system that recapitulates VSG switching. The data indicate that a spontaneous double-stranded DNA break upstream of the gene encoding the coat protein initiates the process. Sleeping sickness is caused by the parasite Trypanosoma brucei. This parasite outwits the human immune system by periodically changing its coat protein in a process known as VSG switching. Here, the first in vitro system that recapitulates VSG switching is established, indicating that a spontaneous double-stranded DNA break upstream of the gene encoding the code protein initiates the process. Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10-2–10-3 per population doubling in recent isolates from nature, but at a markedly reduced rate (10-5–10-6) in laboratory-adapted strains1,2,3. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the ∼70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro ∼250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication.

An efficient method for making directed DNA sequence modifications to plant genes (gene targeting) is at present lacking, thereby frustrating efforts to dissect plant gene function and engineer crop plants that better meet the world's burgeoning need for food, fibre and fuel. Zinc-finger nucleases (ZFNs)-enzymes engineered to create DNA double-strand breaks at specific loci-are potent stimulators of gene targeting; for example, they can be used to precisely modify engineered reporter genes in plants. Here we demonstrate high-frequency ZFN-stimulated gene targeting at endogenous plant genes, namely the tobacco acetolactate synthase genes (ALS SuRA and SuRB), for which specific mutations are known to confer resistance to imidazolinone and sulphonylurea herbicides. Herbicide-resistance mutations were introduced into SuR loci by ZFN-mediated gene targeting at frequencies exceeding 2% of transformed cells for mutations as far as 1.3 kilobases from the ZFN cleavage site. More than 40% of recombinant plants had modifications in multiple SuR alleles. The observed high frequency of gene targeting indicates that it is now possible to efficiently make targeted sequence changes in endogenous plant genes.

Break-induced replication (BIR) is an efficient homologous recombination process to initiate DNA replication when only one end of a chromosome double-strand break shares homology with a template. BIR is thought to re-establish replication at stalled and broken replication forks and to act at eroding telomeres in cells that lack telomerase in pathways known as 'alternative lengthening of telomeres' (reviewed in refs 2, 6). Here we show that, in haploid budding yeast, Rad51-dependent BIR induced by HO endonuclease requires the lagging strand DNA Polalpha-primase complex as well as Poldelta to initiate new DNA synthesis. Polepsilon is not required for the initial primer extension step of BIR but is required to complete 30 kb of new DNA synthesis. Initiation of BIR also requires the nonessential DNA Poldelta subunit Pol32 primarily through its interaction with another Poldelta subunit, Pol31. HO-induced gene conversion, in which both ends of a double-strand break engage in homologous recombination, does not require Pol32. Pol32 is also required for the recovery of both Rad51-dependent and Rad51-independent survivors in yeast strains lacking telomerase. These results strongly suggest that both types of telomere maintenance pathways occur by recombination-dependent DNA replication. Thus Pol32, dispensable for replication and for gene conversion, is uniquely required for BIR; this finding provides an opening into understanding how DNA replication re-start mechanisms operate in eukaryotes. We also note that Pol32 homologues have been identified both in fission yeast and in metazoans where telomerase-independent survivors with alternative telomere maintenance have also been identified.

DNA double-strand breaks (DSBs) are potentially lethal lesions that arise spontaneously during normal cellular metabolism, as a consequence of environmental genotoxins or radiation, or during programmed recombination processes. Repair of DSBs by homologous recombination generally occurs by gene conversion resulting from transfer of information from an intact donor duplex to both ends of the break site of the broken chromosome. In mitotic cells, gene conversion is rarely associated with reciprocal exchange and thus limits loss of heterozygosity for markers downstream of the site of repair and restricts potentially deleterious chromosome rearrangements. DSBs that arise by replication fork collapse or by erosion of uncapped telomeres have only one free end and are thought to repair by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). BIR from one of the two ends of a DSB would result in loss of heterozygosity, suggesting that BIR is suppressed when DSBs have two ends so that repair occurs by the more conservative gene conversion mechanism. Here we show that BIR can occur by several rounds of strand invasion, DNA synthesis and dissociation. We further show that chromosome rearrangements can occur during BIR if dissociation and reinvasion occur within dispersed repeated sequences. This dynamic process could function to promote gene conversion by capture of the displaced invading strand at two-ended DSBs to prevent BIR.

Rad54, a translocase that uses the energy of ATP hydrolysis to move along a duplex, promotes Holliday junction branch migration. Genetic studies in yeast have implicated Rad54 as acting late in recombination, when Holliday junctions are present. Homologous recombination has a crucial function in the repair of DNA double-strand breaks and in faithful chromosome segregation1,2,3. The mechanism of homologous recombination involves the search for homology and invasion of the ends of a broken DNA molecule into homologous duplex DNA to form a cross-stranded structure, a Holliday junction (HJ)4,5,6,7. A HJ is able to undergo branch migration along DNA, generating increasing or decreasing lengths of heteroduplex. In both prokaryotes and eukaryotes, the physical evidence for HJs, the key intermediate in homologous recombination, was provided by electron microscopy8. In bacteria there are specialized enzymes that promote branch migration of HJs7. However, in eukaryotes the identity of homologous recombination branch-migration protein(s) has remained elusive. Here we show that Rad54, a Swi2/Snf2 protein9, binds HJ-like structures with high specificity and promotes their bidirectional branch migration in an ATPase-dependent manner. The activity seemed to be conserved in human and yeast Rad54 orthologues. In vitro, Rad54 has been shown to stimulate DNA pairing of Rad51, a key homologous recombination protein10,11,12. However, genetic data indicate that Rad54 protein might also act at later stages of homologous recombination, after Rad51 (ref. 13). Novel DNA branch-migration activity is fully consistent with this late homologous recombination function of Rad54 protein.

We present a global comparison of differences in content of segmental duplication between human and chimpanzee, and determine that 33% of human duplications (> 94% sequence identity) are not duplicated in chimpanzee, including some human disease-causing duplications. Combining experimental and computational approaches, we estimate a genomic duplication rate of 4-5 megabases per million years since divergence. These changes have resulted in gene expression differences between the species. In terms of numbers of base pairs affected, we determine that de novo duplication has contributed most significantly to differences between the species, followed by deletion of ancestral duplications. Post-speciation gene conversion accounts for less than 10% of recent segmental duplication. Chimpanzee-specific hyperexpansion (> 100 copies) of particular segments of DNA have resulted in marked quantitative differences and alterations in the genome landscape between chimpanzee and human. Almost all of the most extreme differences relate to changes in chromosome structure, including the emergence of African great ape subterminal heterochromatin. Nevertheless, base per base, large segmental duplication events have had a greater impact (2.7%) in altering the genomic landscape of these two species than single-base-pair substitution (1.2%).

A fundamental tenet of classical mendelian genetics is that allelic information is stably inherited from one generation to the next, resulting in predictable segregation patterns of differing alleles. Although several exceptions to this principle are known, all represent specialized cases that are mechanistically restricted to either a limited set of specific genes (for example mating type conversion in yeast) or specific types of alleles (for example alleles containing transposons or repeated sequences). Here we show that Arabidopsis plants homozygous for recessive mutant alleles of the organ fusion gene HOTHEAD (HTH) can inherit allele-specific DNA sequence information that was not present in the chromosomal genome of their parents but was present in previous generations. This previously undescribed process is shown to occur at all DNA sequence polymorphisms examined and therefore seems to be a general mechanism for extra-genomic inheritance of DNA sequence information. We postulate that these genetic restoration events are the result of a template-directed process that makes use of an ancestral RNA-sequence cache.

Whole-genome duplication followed by massive gene loss and specialization has long been postulated as a powerful mechanism of evolutionary innovation. Recently, it has become possible to test this notion by searching complete genome sequence for signs of ancient duplication. Here, we show that the yeast Saccharomyces cerevisiae arose from ancient whole-genome duplication, by sequencing and analysing Kluyveromyces waltii, a related yeast species that diverged before the duplication. The two genomes are related by a 1:2 mapping, with each region of K. waltii corresponding to two regions of S. cerevisiae, as expected for whole-genome duplication. This resolves the long-standing controversy on the ancestry of the yeast genome, and makes it possible to study the fate of duplicated genes directly. Strikingly, 95% of cases of accelerated evolution involve only one member of a gene pair, providing strong support for a specific model of evolution, and allowing us to distinguish ancestral and derived functions.

The male-specific region of the Y chromosome, the MSY, differentiates the sexes and comprises 95% of the chromosome's length. Here, we report that the MSY is a mosaic of heterochromatic sequences and three classes of euchromatic sequences: X-transposed, X-degenerate and ampliconic. These classes contain all 156 known transcription units, which include 78 protein-coding genes that collectively encode 27 distinct proteins. The X-transposed sequences exhibit 99% identity to the X chromosome. The X-degenerate sequences are remnants of ancient autosomes from which the modern X and Y chromosomes evolved. The ampliconic class includes large regions (about 30% of the MSY euchromatin) where sequence pairs show greater than 99.9% identity, which is maintained by frequent gene conversion (non-reciprocal transfer). The most prominent features here are eight massive palindromes, at least six of which contain testis genes.

After gene rearrangement, immunoglobulin variable genes are diversified by somatic hypermutation or gene conversion, whereas the constant region is altered by class-switch recombination. All three processes depend on activation-induced cytidine deaminase (AID)1,2,3,4,5,6,7, a B-cell-specific protein that has been proposed (because of sequence homology1) to function by RNA editing. But indications that the three gene diversification processes might be initiated by a common type of DNA lesion8,9,10,11, together with the proposal that there is a first phase of hypermutation that targets dC/dG12, suggested to us that AID may function directly at dC/dG pairs. Here we show that expression of AID in Escherichia coli gives a mutator phenotype that yields nucleotide transitions at dC/dG in a context-dependent manner. Mutation triggered by AID is enhanced by a deficiency of uracil-DNA glycosylase, which indicates that AID functions by deaminating dC residues in DNA. We propose that diversification of functional immunoglobulin genes is triggered by AID-mediated deamination of dC residues in the immunoglobulin locus with the outcome—that is, hypermutation phases 1 and 2, gene conversion or switch recombination—dependent on the way in which the initiating dU/dG lesion is resolved.

The faithful repair of DNA damage such as chromosomal double-strand breaks (DSBs) is crucial for genomic integrity. Aberrant repair of these lesions can result in chromosomal rearrangements, including translocations, which are associated with numerous tumours. Models predict that some translocations arise from DSB-induced recombination in differentiating lymphoid cell types or from aberrant repair of DNA damage induced by irradiation or other agents; however, a genetic system to study the aetiology of these events has been lacking. Here we use a mouse embryonic stem cell system to examine the role of DNA damage on the formation of translocations. We find that two DSBs, each on different chromosomes, are sufficient to promote frequent reciprocal translocations. The results are in striking contrast with interchromosomal repair of a single DSB in an analogous system in which translocations are not recovered. Thus, while interchromosomal DNA repair does not result in genome instability per se, the presence of two DSBs in a single cell can alter the spectrum of repair products that are recovered.

Patchwork homology observed between divergent members of polymorphic multigene families is thought to reflect evolution by short-tract gene conversion (nonreciprocal recombination), although this mechanism cannot usually be confirmed in higher organisms. In contrast to meiotic conversions observed in laboratory yeast strains, apparent conversions between polymorphic sequences, such as the class I loci of the major histocompatibility complex (MHC), are short and do not seem to be associated with reciprocal recombination (crossover, exchanges). We have now integrated two nonallelic murine class I genes into yeast to characterize their meiotic recombination. We found no crossovers between the MHC genes, but short-tract 'microconversions' of 1-215 base-pairs were observed in about 6% of all meioses. Strikingly, one of these events was accompanied by a single base-pair mutation. These results underscore both the importance of meiotic gene conversion and sequence heterology in determining conversion patterns between divergent genes.

IN Saccharomyces cerevisiae, HO endonuclease-induced mating-type (MAT) switching is a specialized mitotic recombination event in which MAT sequences are replaced by those copied from a distant, unexpressed donor (HML or HMR1,2. The donors have a chromatin structure inaccessible for both transcription and HO cleavage1,2. Here we use physical monitoring of DNA to show that MAT switching is completely blocked at an early step in recombination in strains deleted for the DNA repair genes RAD51, RAD52, RAD54, RAD55 or RAD57. We find, however, that only RAD52 is required when the donor sequence is simultaneously not silenced and located on a plasmid. RAD51, RAD54, RAD55 and RAD57 are still required when the same transcribed donor is on the chromosome. We conclude that recombination in vivo occurs between DNA molecules in chromatin, whose structure significantly influences the outcome. RAD51, RAD54, RAD55 and RAD57 are all required to facilitate strand invasion into otherwise inaccessible donor sequences.

INTRON mobility at the RNA level1& ndash;8 by splicing reversal at allelic (homing) and non-allelic locations (transposition) has been reported in vitro9& ndash;12. In the living cell, however, only intron homing by unidirectional gene conversion has been described5,6,13,14. Supposing that intron insertions at non-allelic sites might occur in vivo, we speculated that group II splice-site-associated macro-deletions15& ndash;18 in fungal mitochondrial DNA might result from group II intron transposition to new locations followed by recombination. We used polymerase chain reaction techniques to detect this critical, infrequent intermediate in mtDNA populations. Here we report on group II intron al1 transposition to non-allelic, splicing-compatible locations within the cox1 gene of yeast mtDNA. The identified integration sites are preceded by motifs similar to the upstream exon A1. Sequences flanking intron al1 are not co-converted to the insertion sites and cis- and trans-acting mutations within al1 reduce intron mobility below detection levels. These findings suggest the involvement of an RNA intermediate in group II intron transposition.

RECOMBINATION between a diffusible reverse transcript and its homologous chromosomal allele has been proposed as a mechanism for the precise removal of introns from DNA and gene conversion of dispersed repeated sequences1,2. We have reported that RNA-mediated recombination occurs in the yeast Saccharomyces cerevisiae3. This recombination requires expression of the retrotransposon Ty, and results in intron loss from a plasmid-borne marker gene and the formation of pseudogenes. Because the pseudogenes are embedded in Ty sequences, chromosomal insertion could have been mediated by Ty integrase or by homologous recombination with endogenous Ty sequences. The structure of the chromosomal recombinants and the fact that plasmid and chromosomal recombination can have different requirements demanded a direct demonstration of RNA-mediated gene conversion of a chromosomal allele. Here we report the first demonstration, to our knowledge, of recombination between a reverse transcript and its chromosomal homologue and describe an assay that specifically detects this novel recombination pathway.

An unusual protein splicing reaction joins the N-terminal segment (A) and the C-terminal segment (C) of the 119K primary translation product (ABC) of the yeast VMA1 gene to yield a 69K vacuolar H(+)-ATPase subunit (AC) and an internal 50K polypeptide (B). This 50K protein is a site-specific DNA endonuclease that shares 34% identity with the homothallic switching endonuclease. The site cleaved by the VMA1-derived endonuclease exists in a VMA1 allele that lacks the derived endonuclease segment of the open reading frame. Cleavage at this site only occurs during meiosis and initiates 'homing', a genetic event that converts a VMA1 allele lacking the endonuclease coding sequence into one that contains it.

DNA termini from Tetrahymena and Oxytricha, which bear C4A2 and C4A2 repeats respectively, can support telomere formation in Saccharomyces cerevisiae by serving as substrates for the addition of yeast telomeric C1-3A repeats. Previously, we showed that linear plasmids with 108 base pairs of C4A4DNA (YLp108CA) efficiently acquired telomeres, whereas plasmids containing 28,64 base pairs of C4A4 DNA also promoted telomere formation, but with reduced efficiency1. Although many of the C4A4 termini on these plasmids underwent recombination with a C4A2 terminus, the mechanism of telomere-telomere recombination was not established1,3. We now report the sequence of the C4A4 ends from the linear plasmids. The results provide strong evidence for a novel recombination process involving a gene conversion event that requires little homology, occurs at or near the boundary of telomeric and non-telomeric DNA, and resembles the recombination process involved in bacteriophage T4 DNA replication.

GENETIC recombination involves classical crossing-over and gene conversion (aberrant segregation). In fungi that produce an ascus containing four spores, a gene conversion event is manifested as 3:1 or 1:3 (or more rarely 4:0 or 0:4) segregations, in contrast to the normal mendelian 2:2 segregation1,2. Polarity is one of the properties of gene conversion; in almost all cases the frequency of conversion exhibits a gradient across the gene monitored1. The frequency of conversion is usually independent of the specific allele used as a marker, but dependent on its location. An interpretation of conversion polarity is that it is caused by the existence of specific initiation sites for meiotic recombination, located at the high end of the polarity gradient. Here we show that the polarity gradient for the HIS2 gene of Saccharomyces cerevisiae is high at the 3′ end of the gene, implying that the promoter of HIS2 is not the initiation site.

The nonapeptide hormones arginine vasopressin (AVP) and oxytocin (OT) are synthesized in the hypothalamus together with their carrier proteins, the neurophysins, as common poly-peptide precursors1–4. The organization of these precursors has been established by sequence determination of cloned bovine cDNAs encoding prepro-arginine vasopressin-neurophysin II (prepro-AVP-NPII) and prepro-oxytocin-neurophysin I (prepro-OT-NPI)5,6. When the mRNA sequences coding for the conserved middle part of the neurophysins were compared, we found that these sequences are not merely similar but identical6. The primary structure of the chromosomal genes now determined shows that both genes, which appear to have arisen by a gene duplication, are split into three exons, each encoding a functional domain of the precursor polypeptide. Sequence comparison reveals that the stretch of sequence identity within the two mRNAs is probably the result of a gene conversion encompassing exonB, which encodes the conserved part of the neurophysins, and part of the preceding intron.

The association of reciprocal exchange with intrachromosomal gene conversion has been examined using an inverted repeat at the HIS3 locus of Saccharomyces cerevisiae. Intrachromosomal gene conversions between the duplicated inverted sequences are frequent. In contrast to gene conversion events between homologous chromosomes, intrachromosomal gene conversion is not associated with reciprocal exchange in flanking sequences. This observation has important consequences for the role of gene conversion in the maintenance of multigene families.

Injection of homologous DNA sequences into nuclei of cultured mammalian cells induces mutations in the cognate chromosomal gene. It appears that these mutations result from incorrect repair of a heteroduplex formed between the introduced and the chromosomal sequence. This phenomenon is termed 'heteroduplex induced mutagenesis'. The high frequency of these events suggests that this method may prove useful for introducing mutations into specific mammalian genes.

The mammalian major histocompatibility complex (MHC; H–2 complex in mouse) is a large multigene complex which encodes cell-surface antigens involved in the cellular immune response to foreign antigens1. Class I polypeptides expressed at the H–2K and H–2D (Fig. 1) loci of numerous mouse strains exhibit an unusually high degree of genetic polymorphism, which is assumed to be related to their function as primary recognition elements in the immune response. We suggested that this H–2 polymorphism may arise by gene conversion-like events between non-allelic class I genes. This is supported by our recent comparison of the DNA sequences of the normal H–2Kb gene sequence, from the C57BL/10 mouse, and a mutant2,3 form of this gene called H–2Kbm1 (ref. 4): the mutant allele differs from the H–2Kb gene in seven bases out of a region of 13 bases in exon 3 of the class I gene (which encodes α2 (C1) the second highly polymorphic protein domain), suggesting that this region of new sequence had been introduced into the H–2Kb sequence following unequal pairing of two class I genes in the genome of the C57BL mouse. Schulze et al. have obtained similar results5. Here we report work identifying a potential donor gene in our library of 26 class I genes cloned from the C57BL/10 mouse.

DNA regions which are composed of a single or relatively few short sequence motifs usually in tandem (‘pure simple sequences’) have been reported in the genomes of diverse species (for reviews see refs 1–4), and have been implicated in a range of functions including gene regulation (for reviews see refs 5–7), signals for gene conversion and recombination8–10, and the replication of telomeres11They are thought to accumulate by DNA slippage12–16 and mispairing during replication and recombination or extension of single-strand ends2,4,10,11. In order to systematize the range of DNA simplicity and the genetic nature of the regions that are simple, we have undertaken an extensive computer search of the DNA sequence library of the European Molecular Biology Laboratory (EMBL)17. We show here that nearly all possible simple motifs occur 5–10 times more frequently than equivalent random motifs. Furthermore, a new computer algorithm reveals the widespread occurrence of significantly high levels of a new type of ‘cryptic simplicity’ in both coding and noncoding DNA. Cryptically simple regions are biased in nucleotide composition and consist of scrambled arrangements of repetitive motifs which differ within and between species. The universal existence of DNA simplicity from monotonous arrays of single motifs to variable permutations of relatively short-lived motifs suggests that ubiquitous slippage-like mechanisms are a major source of genetic variation in all regions of the genome, not predictable by the classical mutation process.

Different molecular weight forms of the protein product of the yeast mitochondrial gene var1 are shown to arise by a process of asymmetric gene conversion. These different forms can be accounted for by two DNA segments, ∼36 and 57 base pairs long, present in one allelic form of the var1 structural gene, which can be inserted independently and at different frequencies into other var1 alleles.

The mitotic recombination behaviour of a duplication of the his4 region on chromosome III in the yeast Saccharomyces cerevisiae was studied. The major recombination event between the duplicated segments is gene conversion unassociated with reciprocal recombination. The rad52-1 mutation preferentially decreases mitotic gene conversion. These results suggest that mitotic gene conversion may occur by a different pathway from that occurring in meiosis. This mitotic gene conversion may be important in yeast mating type interconversion and the maintenance of sequence homogeneity in families of repeated eukaryotic genes.

Several species related to Drosophila melanogaster have two loci containing sequences which code for the major heat-shock protein of molecular weight 70,000 (hsp70). These sequences are arranged as two pairs of inverted repeats. The structure of the 87C locus in D. melanogaster seems to have arisen through DNA insertion. Hsp70-coding sequences at the two loci are not evolving independently. Gene conversion between hsp70 genes has apparently occurred both within and between loci.

During meiosis in fission yeast, transfer of genetic information occurs between tRNA genes of related sequence even when they are located on different chromosomes. This process of intergenic conversion has been analysed with the help of nonsense suppressors derived from three unlinked serine tRNA genes. In their wild-type form two of the genes code for tRNASerUCA, the third for tRNASerUCG. We demonstrate the transfer of anticodon and adjacent intron sequences between two tRNA genes coding for different serine isoacceptors.

Many pathogenic bacteria express pili (fimbriae) on their cell surfaces. These structures mediate binding of bacteria to host tissues, and may also be involved in other aspects of pathogenesis. Neisseria gonorrhoeae pili are mainly composed of a single protein, pilin, whose expression is controlled at chromosomal expression loci (pilE). An intact pilin gene and promoter sequences are only found at pilE. Strain MS11 contains two expression sites (pilE1 and pilE2), whereas several of its derivatives and other clinical isolates contain only one. Silent pilin loci (pilS1-pilS7) contain truncated variant pilin genes lacking the promoter and conserved pilin gene sequences. Pilin antigenic variation in N. gonorrhoeae occurs by DNA recombination between one of he silent partial variant gene segments in pilS and an expressed pilin gene in pilE. The recombination reactions are nonreciprocal, and therefore the mechanism has been classified as gene conversion. We report that much of the recombination between pilin loci actually occurs after transformation of living piliated cells by DNA liberated from lysed cells within a population. This constitutes a new molecular mechanism for an antigenic variation system, as well as the first specific function for a DNA transformation system.