The TopoVIB-Like protein family is required for meiotic DNA double-strand break formation.

Meiotic recombination is induced by the formation of DNA double-strand breaks (DSBs) catalyzed by SPO11, the ortholog of subunit A of TopoVI DNA topoisomerase (TopoVIA). TopoVI activity requires the interaction between A and B subunits. We identified a conserved family of plant and animal proteins [the TOPOVIB-Like (TOPOVIBL) family] that share strong structural similarity to the TopoVIB subunit of TopoVI DNA topoisomerase. We further characterize the meiotic recombination proteins Rec102 (Saccharomyces cerevisiae), Rec6 (Schizosaccharomyces pombe), and MEI-P22 (Drosophila melanogaster) as homologs to the transducer domain of TopoVIB. We demonstrate that the mouse TOPOVIBL protein interacts and forms a complex with SPO11 and is required for meiotic DSB formation. We conclude that meiotic DSBs are catalyzed by a complex involving SPO11 and TOPOVIBL.

The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis.

Spermatogenesis relies on the precise regulation of the self-renewal and differentiation of spermatogonia to provide a continuous supply of differentiating germ cells. The understanding of the cellular pathways regulating this equilibrium remains unfortunately incomplete. This investigation aimed to elucidate the testicular and ovarian functions of the glucocorticoid-induced leucine zipper protein (GILZ) encoded by the X-linked Tsc22d3 (Gilz) gene. We found that GILZ is specifically expressed in the cytoplasm of proliferating spermatogonia and preleptotene spermatocytes. While Gilz mutant female mice were fully fertile, constitutive or male germ cell-specific ablation of Gilz led to sterility due to a complete absence of post-meiotic germ cells and mature spermatozoa. Alterations were observed as early as postnatal day 5 during the first spermatogenic wave and included extensive apoptosis at the spermatogonial level and meiotic arrest in the mid-late zygotene stage. Overall, these data emphasize the essential role played by GILZ in mediating spermatogonial survival and spermatogenesis.

PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice.

Meiotic recombination events cluster into narrow segments of the genome, defined as hotspots. Here, we demonstrate that a major player for hotspot specification is the Prdm9 gene. First, two mouse strains that differ in hotspot usage are polymorphic for the zinc finger DNA binding array of PRDM9. Second, the human consensus PRDM9 allele is predicted to recognize the 13-mer motif enriched at human hotspots; this DNA binding specificity is verified by in vitro studies. Third, allelic variants of PRDM9 zinc fingers are significantly associated with variability in genome-wide hotspot usage among humans. Our results provide a molecular basis for the distribution of meiotic recombination in mammals, in which the binding of PRDM9 to specific DNA sequences targets the initiation of recombination at specific locations in the genome.

Identification and characterization of an SPO11 homolog in the mouse.

The SPO11/TOPVIA family includes proteins from archaebacteria and eukaryotes. The protein member from the archaebacterium Sulfulobus shibatae is the catalytic subunit of TopoVI DNA topoisomerase. In Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, SPO11 is required for meiotic recombination, suggesting a conserved mechanism for the initiation step of this process. Indeed, S. cerevisiae SPO11 has been shown to be directly involved in the formation of meiotic DNA double-strand breaks that initiate meiotic recombination. Here, we report the identification of a Mus musculus Spo11 cDNA, which encodes a protein closely related to all members of the SPO11/TOPVIA family. cDNAs resulting from alternative splicing were detected, suggesting that there are potential variants of the mouse SPO11 protein. By RNA-blotting analysis, expression of the mouse Spo11 gene was detected only in the testis, in agreement with its predicted function in the initiation of meiotic recombination. We mapped the mouse Spo11 gene to chromosome 2, band H2-H4.

The essential role of yeast topoisomerase III in meiosis depends on recombination.

Yeast cells mutant for TOP3, the gene encoding the evolutionary conserved type I-5' topoisomerase, display a wide range of phenotypes including altered cell cycle, hyper-recombination, abnormal gene expression, poor mating, chromosome instability and absence of sporulation. In this report, an analysis of the role of TOP3 in the meiotic process indicates that top3Delta mutants enter meiosis and complete the initial steps of recombination. However, reductional division does not occur. Deletion of the SPO11 gene, which prevents recombination between homologous chromosomes in meiosis I division, allows top3Delta mutants to form viable spores, indicating that Top3 is required to complete recombination successfully. A topoisomerase activity is involved in this process, since expression of bacterial TopA in yeast top3Delta mutants permits sporulation. The meiotic block is also partially suppressed by a deletion of SGS1, a gene encoding a helicase that interacts with Top3. We propose an essential role for Top3 in the processing of molecules generated during meiotic recombination.

[Mechanisms and control of meiotic recombination in the yeast Saccharomyces cerevisiae]. / Mécanismes et contrôles de la recombinaison méiotique chez la levure Saccharomyces cerevisiae.

Recent studies in Saccharomyces cerevisiae have provided new insights in our understanding of the molecular mechanisms of meiotic recombination. Meiosis-specific DNA double-strand breaks have been detected and have been shown to be the lesions that initiate recombination events. These are located mostly in promoter regions where the chromatin is in an open configuration, and cluster in domains along the chromosome. They are likely to be made by a topoisomerase II-like protein encoded by the SPO11 gene. Several DNA intermediates in the meiotic double strand-break repair pathway have been characterised and several multi-protein complexes have been identified and shown to be involved at different steps in the repair pathway. The conservation of these protein complexes in higher eukaryotes suggests that the meiotic recombination mechanism could be conserved. With the application of the well characterised genetical, molecular, cytological and biochemical techniques and the recently developed technology for genomic studies (biochips), we can expect a rapid increase in our comprehension of the meiotic recombination process.

Mitotic recombination and localized DNA double-strand breaks are induced after 8-methoxypsoralen and UVA irradiation in Saccharomyces cerevisiae.

Mitotic recombination within the ARG4 gene of Saccharomyces cerevisiae was analysed after treatment of cells with the recombinogenic agent 8-methoxypsoralen (8-MOP) plus UVA. The appearance of DNA double-strand breaks (DSBs) in the ARG4 region during post-treatment incubation was also tested. The results obtained after 8-MOP plus UVA treatment indicate that in mitotic cells: (1) recombination at the ARG4 locus is increased 30 - 500 fold per survivor depending on the strains and the doses employed, (2) the increase of recombination results essentially from gene conversion events which involve the RV site located in the 5' region of the ARG4 gene twice as often as the Bgl site at the 3' end, (3) depending on 8-MOP/UVA dose, ectopic gene conversion is associated with reciprocal translocation, (4) DSBs occur preferentially in the ARG 5' region during post-treatment incubation, as well as in other intergenic regions containing both promoters or/and terminators of transcription, and (5) changes in sequence content in the 5' region of ARG4, which influences positions and frequencies of DSBs formed during repair, are correlated with a modification of the local chromatin structure.

An atypical topoisomerase II from Archaea with implications for meiotic recombination.

Type II topoisomerases help regulate DNA topology during transcription, replication and recombination by catalysing DNA strand transfer through transient double-stranded breaks. All type II topoisomerases described so far are members of a single protein family. We have cloned and sequenced the genes encoding the A and B subunits of topoisomerase II from the archaeon Sulfolobus shibatae. This enzyme is the first of a new family. It has no similarity with other type II topoisomerases, except for three motifs in the B subunit probably involved in ATP binding and hydrolysis. We also found these motifs in proteins of the Hsp90 and MutL families. The A subunit has similarities with four proteins of unknown function. One of them, the Saccharomyces cerevisiae Spo11 protein, is required for the initiation of meiotic recombination. Mutagenesis, performed on SPO11, of the single tyrosine conserved between the five homologues shows that this amino acid is essential for Spo11 activity. By analogy with the mechanism of action of known type II topoisomerases, we suggest that Spo11 catalyses the formation of double-strand breaks that initiate meiotic recombination in S. cerevisiae.

The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae.

Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae occurs by localized DNA double-strand breaks (DSBs) at several locations in the genome, corresponding to hot spots for meiotic gene conversion and crossing over. The meiotic DSBs occur in regions of chromatin that are hypersensitive to nucleases. To gain insight into the molecular mechanism involved in the formation of these DSBs, we have determined their positions at the nucleotide level at the CYS3 hot spot of gene conversion on chromosome I. We found four major new features of these DSBs: (i) sites of DSBs are multiple with varying intensities and spacing within the promoter region of the CYS3 gene; (ii) no consensus sequence can be found at these sites, indicating that the activity involved in DSB formation has little or no sequence specificity; (iii) the breaks are generated by blunt cleavages; and (iv) the 5' ends are modified in rad50S mutant strains, where the processing of these ends is known to be prevented. We present a model for the initiation of meiotic recombination taking into account the implications of these results.

Initiation of recombination in Saccharomyces cerevisiae haploid meiosis.

In most eukaryotes during prophase I of meiosis, homologous chromosomes pair and recombine by coordinated molecular and cellular processes. To directly test whether or not the early steps of the initiation of recombination depend on the presence of a homologous chromosome, we have examined the formation and processing of DNA double-strand breaks (DSBs, the earliest physical landmark of recombination initiation) in various haploid Saccharomyces cerevisiae strains capable of entering meiosis. We find that DSBs occur in haploid meiosis, showing that the presence of a homolog is not required for DSB formation. DSBs occur at the same positions in haploid and diploid meioses. However, these two types of meiosis exhibit subtle differences with respect to the timing of formation and levels of DSBs. In haploid meiosis, a slower rate of DSB formation and a reduction in the frequency of DSB (at one of the three sites analyzed) were observed. These results might indicate that interactions between homologs play a role in the formation of meiotic DSBs. Furthermore, haploid strains exhibit a pronounced delay in the disappearance of meiotic DSBs compared to diploid strains, which suggests that sister chromatid interactions for DSB repair are inhibited in haploid meiosis.

The control in cis of the position and the amount of the ARG4 meiotic double-strand break of Saccharomyces cerevisiae.

During meiosis, a transient DNA double-strand break (DSB) occurs in the promoter region (positions -200/-185) of the Saccharomyces cerevisiae ARG4 gene and is a likely intermediate in the initiation of meiotic gene conversion events in this region. We report here a functional analysis of the ARG4 DSB based on the study of various deletions in this chromosomal region. We have identified several cis-acting elements located within the -465/+3 region of the ARG4 promoter that control the formation of this DSB. The -465/-317 region includes a transcription terminator and is necessary for a normal amount of ARG4 DSB, but not for its positioning. The -316/-140 region can be replaced by an unrelated DNA sequence where a meiotic DSB then occurs, suggesting that the site of DSB is not sequence-specific, but is positioned at a fixed distance from the adjacent -139/+3 region. Also, in all strains constructed, the amount of meiotic DSB is correlated with the frequency of gene conversion in ARG4, which provides a strong argument for the initiation of gene conversion by a DSB in this region of the yeast genome.

The Saccharomyces cerevisiae ARG4 initiator of meiotic gene conversion and its associated double-strand DNA breaks can be inhibited by transcriptional interference.

In the yeast Saccharomyces cerevisiae, as in other eukaryotes, some regions of the genome have a much higher level of meiotic gene conversion than others. Previous deletion analysis indicated that the sequence necessary for the high level of gene conversion within the ARG4 region defined an initiation site located between positions -316 and -37 [relative to the first base pair (+1) of the ARG4 coding sequence] of the ARG4 promoter. To test whether this sequence is sufficient to promote gene conversion in a novel chromosomal context, we inverted on the chromosome various DNA fragments including the implicated region and the ARG4 coding sequence. Surprisingly, these inversions resulted in the loss of the normal recombination properties and double-strand-break formation associated with this process. By Northern analysis, we found that a transcript traverses the ARG4 initiation site in these inversion mutants but not in the wild type. When transcription through this region was prevented by a transcription terminator, the activity of the initiation site and the formation of double-strand breaks were restored. From these results and from complementary deletion analysis in the normal ARG4 orientation, we conclude that the activity of the ARG4 initiation site requires protection from transcriptional interference.

Mutations of the phage lambda attachment site alter the directionality of resolution of Holliday structures.

Integrative recombination of bacteriophage lambda occurs by two sequential, reciprocal strand exchanges at specific positions within the attachment sites. Both exchanges are promoted by the lambda Int protein; the first forms a Holliday structure, and the second resolves it to recombinant products. Recombination requires sequence homology within the 7 bp 'overlap' region that separates the two points of strand exchange. To see if homology promotes the second strand exchange, we constructed attachment site Holliday structures by annealing DNA strands and then assayed Int-promoted resolution. Holliday structures corresponding to strand exchange between sites with homologous overlap regions were efficiently resolved to give mixtures of recombinants and parents. Holliday structures corresponding to exchanges between heterologous sites fell into two classes. Members of the first class, in which heterology limited but did not completely prevent migration of the branchpoint within the overlap region, were resolved efficiently and preferentially to parental molecules. We propose that resolution to recombinants occurs only if homology allows branch migration from the first to the second exchange site. Members of the second class, in which heterology constrained the branchpoint within an Int binding site, were resolved poorly. We suggest that Holliday structures that have a branchpoint within an Int binding site are poor substrates for Int.

Inhibition of replication forks exiting the terminus region of the Escherichia coli chromosome occurs at two loci separated by 5 min.

The replication cycle of Escherichia coli strains duplicating their chromosome from the same plasmid origin placed at various locations or of strains having undergone a major inversion event along the origin-to-terminus axis was studied by marker-frequency analysis. It was observed that replication forks are unidirectionally inhibited at two loci of the termination region: counterclockwise-moving forks are inhibited at terminator T1 (28.5 min), and forks moving in the opposite direction are inhibited at terminator T2 (33.5 min). By determining the strand preference of Okazaki fragments that are specific for markers from the T1-T2 interval, it was shown that this interval is replicated in either direction, depending upon the strain analyzed. In addition, we also observed that forks moving in the "unnatural" direction along each oriC-T1 or -T2 arm are very slow, especially in the one-third portion of the chromosome around the terminators. We propose that this phenomenon is a consequence of nucleoid organization, which is proposed to be symmetrical on the two oriC-T1 or -T2 arms and polarized with respect to the direction of replication. We also propose that T1 and T2 are the terminal limits of these two polarized half-nucleoid bodies.

Gene 3 endonuclease of bacteriophage T7 resolves conformationally branched structures in double-stranded DNA.

Gene 3 endonuclease of bacteriophage T7 has been expressed from the cloned gene, purified, and characterized as to its activity on different DNA substrates. Besides its known strong preference for cutting single-stranded DNA rather than double-stranded DNA, the enzyme has a strong preference for cutting conformationally branched structures in double-stranded DNA, either X or Y-shaped branches. Three types of branched DNA substrates were used: relaxed circular DNAs containing large cruciform structures (a model for Holliday structures, presumed intermediates in genetic recombination); X-shaped molecules having a limited potential for branch migration, made from the cloned phage and bacterial arms of the lambda attachment site; and Y-shaped molecules, made by hybridizing molecules homologous except for a 2 X 21 base-pair palindrome in one of them. Gene 3 endonuclease cuts two opposing strands at or near the branchpoint to resolve these substrates into linear molecules, and does not cut the potentially single-stranded tips of the stem-and-loop structure generated from the palindrome. The position of the cleavage points on the equivalent arm of two X-shaped molecules, constructed from wild-type and mutant lambda attachment sites, show that the enzyme can cut at several different sites within or slightly 5' of the limited region of branch migration. The various activities of gene 3 endonuclease are consistent with the known role of this enzyme in genetic recombination, in maturation and packaging of T7 DNA, and in degradation of host DNA, and suggest that the enzyme recognizes a specific structural feature in DNA. Its cleavage specificity, ready availability, and ability to act at physiological pH and ionic conditions may make gene 3 endonuclease useful as a probe for specific DNA structures or for binding of proteins that alter DNA structure.

Multiple origin usage for DNA replication in sdrA(rnh) mutants of Escherichia coli K-12. Initiation in the absence of oriC.

In stable DNA replication (sdrA/rnh) mutants of Escherichia coli, initiation of rounds of DNA replication occurs in the absence of the normal origin of replication, oriC. To determine whether or not the initiation occurs at a fixed site(s) on the chromosome in sdrA mutants, the DNA from exponentially growing sdrA mutant cells with or without the oriC site (delta oriC) was analyzed for the relative copy numbers of various genes along the chromosome. The results suggest that there are at least four fixed sites or regions of the sdrA delta oriC chromosome from which DNA replication can be initiated in the absence of the oriC sequence.

oriX: a new replication origin in E. coli.

Replication of the chromosome of E. coli at 42 degrees C in an integratively suppressed dnaA mutant (dnaA46 Sin Hfr) occurs predominantly from the origin of replication of the integrated plasmid (oriV). We have carried out a detailed marker frequency analysis on such Hfrs. This analysis indicates that replication at 42 degrees C occurs not only from oriV, but also from an origin, oriX, located in the terminal region of the chromosome close to, but distinct from, the prophage rac (oriJ). In an oxa1 mutant of one of these Hfrs, we have shown that replication proceeds at 42 degrees C from all three origins: oriV, oriX, and oriC. Loss of the integrated plasmid results in a temperature- and rich-medium-sensitive strain that replicates the chromosome from oriC and oriX. Replication from oriX proceeds slowly and bidirectionally. We suggest that oriX may be involved in the coupling between replication and cell division.