Meiotic Homologous Recombination Research Articles

Two variants of MutS homolog hMSH5: Prevalence in humans and effects on protein interaction

MSH5 is known to play functional roles in an array of cellular processes such as DNA damage response and meiotic homologous recombination. Here, we report the characterization of an hMSH5 splicing variant (hMSH5sv) that resulted from the retention of the last 51 bp of hMSH5 intron 6, in which the encoded 17-amino acid insertion between codons 179 and 180 does not compromise its capability to interact with hMSH4. We have also identified an hMSH5 polymorphism (C84T) that altered codon 29 of the hMSH5 gene resulting in a proline-to-serine change (P29S). The interaction domains of hMSH4 and hMSH5 have also been resolved. The P29S alteration is located within the interacting domain and leads to a weakened protein interaction with hMSH4. Together, our present study revealed the existence of two forms of hMSH5 variants in human cells. The different properties associated with these two hMSH5 variants underscore the potential functional diversity of the human hMSH5 gene.

An unusual cosegregation of cmt1a duplication and hnpp PMP22 point mutation in a family with CMT1 disease

The common CMT1A duplication and HNPP deletion arise from unequal crossing over and homologous recombination during meiosis between flanking repeats (CMT1A‐REP). CMT1A and HNPP thus result from an altered copy number of the dosage‐sensitive myelin gene PMP22 which is included in the duplicated/deleted tract. However, both diseases can also be caused by rare PMP22 point mutations. We report here a unique family carrying both the CMT1A duplication and an HNPP PMP22 point mutation. A CMT1 child (III‐1), the only affected member of her family, was found to carry the duplication. Surprisingly, the mutation was also found in the mother (II‐2) who had normal clinical and electrophysiologic phenotype. PMP22 sequence analysis in both individuals uncovered a truncating frameshift mutation (Leu145fs) in the healthy mother but not in the affected child. In the mother, this mutation, which had been previously identified by us in 3 other HNPP families, would inactivate the extra copy of the PMP22 gene, functionally compensating the effects of the duplication. Further investigations in the family demonstrated that the healthy maternal grandmother (I‐1) also carried both the duplication and the point mutation, while her other son (II‐1) had neither of them, which indicates that both mutations were harbored by the same chromosome. Microsatellite analysis suggests that a meiotic homologous recombination event has occurred in the affected child, replacing the mutated PMP22 copy with a functional one and resulting in CMT1 phenotype.

Characterization of a DMC1 homologue, RiLIM15, in meiotic panicles, mitotic cultured cells and mature leaves of rice (Oryzasativa L.)

DMC1 is one of the most important genes involved in meiotic homologous recombination in Saccharomyces cerevisiae. Homologues of DMC1 have been isolated recently from some plant species, and in this study, we characterized the structure and expression of a DMC1 homologue, RiLIM15, in a Japonica rice, strain A58. RiLIM15 was found to be a gene family consisting of two genes, RiLIM15A and RiLIM15B, in the rice genome. The DNA sequence of RiLIM15A was highly homologous with that of RiLIM15B in the exon regions, although it was less homologous with that of RiLIM15B in the intron regions. Analysis for the expression of RiLIM15 by a combination of Southern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) showed that RiLIM15 was expressed not only in meiotic young panicles, but also in mitotic cultured cells, although not in the mature leaves. Analysis of the sequences of these RiLIM15 cDNAs amplified by RT-PCR showed that the sequences of exon 5 were deleted from the cDNAs derived from the meiotic young panicles. Also, exons 5, 10 and 11, as well as 29 bp of exon 8, were deleted from some types of cDNA from the mitotic cultured cells. These results suggest that these deletions may be caused by alternative splicing.

Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs.

The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.

Hyper-resistance of meiotic cells to radiation due to a strong expression of a single recA-like gene in Caenorhabditis elegans

Sensitivity of meiotic cells to DNA damaging agents is little understood. We have demonstrated that the meiotic pachytene nuclei in the Caenorhabditis elegans gonad are hyper-resistant to X-ray irradiation, but not to UV irradiation, whereas the early embryonic cells after fertilization and the full grown oocytes are not. The Ce-rdh-1 gene [RAD51, DMC1 (LIM15), homolog 1 or Ce-rad-51], which is essential for the meiotic recombination, is the only bacterial recA-like gene in the nematode genome, and is strongly expressed in the meiotic cells. Following silencing of the Ce-rdh-1 gene by RNA interference, the meiotic cells become more sensitive to X-ray irradiation than the early embryonic cells. This is the first report that meiotic cells are hyper-resistant to DNA strand breaks due to the high level of expression of the enzyme(s) involved in meiotic homologous recombination.

The Rad51 paralog Rad51B promotes homologous recombinational repair.

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B(-/-) cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B(-/-) cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to gamma-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B(-/-) cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.

Effect of the topoisomerase-II inhibitor etoposide on meiotic recombination in male mice

Unlike other chemicals that have been tested in mammalian germ cells, the type-II topoisomerase inhibitor etoposide exhibits significant mutagenicity in primary spermatocytes. Because this is the cell stage during which meiotic recombination normally occurs, and because topoisomerases play a role in recombination, we studied the effect of etoposide on crossing-over in male mice. Exposure to those meiotic prophase stages (probably early to mid-pachytene) during which specific-locus deletion mutations can be induced resulted in decreased crossing-over in the p-Tyr(c) interval of mouse chromosome 7. Accompanying cytological studies with fluorescent antibodies indicated that while there was no detectable effect on the number of recombination nodules (MLH1 foci), there were marked changes in the stage of appearance and localization of RAD51 and RPA proteins. These temporal and spatial protein patterns suggest the formation of multiple lesions in the DNA after MLH1 has already disappeared from spermatocytes. Since etoposide blocks religation of the cut made by type II topoisomerases, repair of DNA damage may result in rejoining of the original DNA strands, undoing the reciprocal exchange that had already occurred and resulting in reduced crossing-over despite a normal frequency of MLH1 foci. Crossing-over could conceivably be affected differentially in different chromosomal regions. If, however, the predominant action of etoposide is to decrease homologous meiotic recombination, the chemical could be expected to increase nondisjunction, an event associated with human genetic risk. Three periods in spermatogenesis respond to etoposide in different ways. Exposure of (a) late differentiating spermatogonia (and, possibly, preleptotene spermatocytes) results in cell death; (b) early- to mid-pachytene induces specific-locus deletions and crossover reduction; and, (c) late pachytene-through-diakinesis leads to genetically unbalanced conceptuses as a result of clastogenic damage.

Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis.

Double-strand DNA breaks (DSBs) pose a major threat to living cells, and several mechanisms for repairing these lesions have evolved. Eukaryotes can process DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ). NHEJ connects DNA ends irrespective of their sequence, and it predominates in mitotic cells, particularly during G1 (ref. 3). HR requires interaction of the broken DNA molecule with an intact homologous copy, and allows restoration of the original DNA sequence. HR is active during G2 of the mitotic cycle and predominates during meiosis, when the cell creates DSBs (ref. 4), which must be repaired by HR to ensure proper chromosome segregation. How the cell controls the choice between the two repair pathways is not understood. We demonstrate here a physical interaction between mammalian Ku70, which is essential for NHEJ (ref. 5), and Mre11, which functions both in NHEJ and meiotic HR (Refs 2,6). Moreover, we show that irradiated cells deficient for Ku70 are incapable of targeting Mre11 to subnuclear foci that may represent DNA-repair complexes. Nevertheless, Ku70 and Mre11 were differentially expressed during meiosis. In the mouse testis, Mre11 and Ku70 co-localized in nuclei of somatic cells and in the XY bivalent. In early meiotic prophase, however, when meiotic recombination is most probably initiated, Mre11 was abundant, whereas Ku70 was not detectable. We propose that Ku70 acts as a switch between the two DSB repair pathways. When present, Ku70 destines DSBs for NHEJ by binding to DNA ends and attracting other factors for NHEJ, including Mre11; when absent, it allows participation of DNA ends and Mre11 in the meiotic HR pathway.

Human Meiotic Recombination Products Revealed by Sequencing a Hotspot for Homologous Strand Exchange in Multiple HNPP Deletion Patients

The HNPP (hereditary neuropathy with liability to pressure palsies) deletion and CMT1A (Charcot-Marie-Tooth disease type 1A) duplication are the reciprocal products of homologous recombination events between misaligned flanking CMT1A-REP repeats on chromosome 17p11. 2-p12. A 1.7-kb hotspot for homologous recombination was previously identified wherein the relative risk of an exchange event is 50 times higher than in the surrounding 98.7% identical sequence shared by the CMT1A-REPs. To refine the region of exchange further, we designed a PCR strategy to amplify the recombinant CMT1A-REP from HNPP patients as well as the proximal and distal CMT1A-REPs from control individuals. By comparing the sequences across recombinant CMT1A-REPs to that of the proximal and distal CMT1A-REPs, the exchange was mapped to a 557-bp region within the previously identified 1.7-kb hotspot in 21 of 23 unrelated HNPP deletion patients. Two patients had recombined sequences suggesting an exchange event closer to the mariner-like element previously identified near the hotspot. Five individuals also had interspersed patches of proximal or distal repeat specific DNA sequence indicating potential gene conversion during the exchange of genetic material. Our studies provide a direct observation of human meiotic recombination products. These results are consistent with the hypothesis that minimum efficient processing segments, which have been characterized in Escherichia coli, yeast, and cultured mammalian cells, may be required for efficient homologous meiotic recombination in humans.

Identification of a Novel HumanRAD51Homolog,RAD51B

The highly conservedSaccharomyces cerevisiaeRAD51 protein functions in both mitotic and meiotic homologous recombination and in double-strand break repair. Screening of the public cDNA sequence database forRAD51-like genes led to the identification of a partial sequence from a breast tissue library present in the I.M.A.G.E. (Integrated Molecular Analysis of Genes and their Expression) collection. An extended 1764-bp cDNA clone encoding an open reading frame of 350 amino acids was isolated. This clone showed significant amino acid identity with other human RAD51 homologs. The new homolog, namedRAD51B,was mapped to human chromosome 14q23–q24.2 using a panel of human–hamster somatic cell hybrids and fluorescencein situhybridization. Northern blot analysis demonstrated thatRAD51BmRNA is widely expressed and most abundant in tissues active in recombination. Functions associated with known RAD51 homologs suggest a role for RAD51B in meiotic recombination and/or recombinational repair.

Germinal excisions of the maize transposon activator do not stimulate meiotic recombination or homology-dependent repair at the bz locus.

Double-strand breaks have been implicated both in the initiation of meiotic recombination in yeast and as intermediates in the transposition process of nonreplicative transposons. Some transposons of this class, notably P of Drosophila and Tc1 of Caenorhabditis elegans, promote a form of homology-dependent premeiotic gene conversion upon excision. In this work, we have looked for evidence of an interaction between Ac transposition and meiotic recombination at the bz locus in maize. We find that the frequency of meiotic recombination between homologues is not enhanced by the presence of Ac in one of the bz heteroalleles and, conversely, that the presence of a homologous sequence in either trans (homologous chromosome) or cis (tandem duplication) does not promote conversion of the Ac insertion site. However, a tandem duplication of the bz locus may be destabilized by the insertion of Ac. We discuss possible reasons for the lack of interaction between Ac excision and homologous meiotic recombination in maize.

2 Meiotic Recombination Hotspots: Shaping the Genome and Insights into Hypervariable Minisatellite DNA Change

Meiotic homologous recombination serves three principal roles. First, recombination reassorts 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 minisatellite DNA repeats.

The nine-parameter gene conversion model: simpler equations, validity tests, and multiple fits

Lamb and Zwolinski (1992, 1993) provided equations and a method for finding nine parameter values for gene conversion between two alleles at one locus, to study mechanisms for homologous meiotic recombination. Simpler, shorter, and easier-to-use equations have been given here, and the accuracy of the parameter values found has been analysed. Fincham's (1994) criticisms of this method have been tested in several ways and shown to be incorrect. The model's validity has been tested directly: sets of arbitrarily chosen parameter values were used to work out the numbers expected in each segregation class for a sample of 100,000 octads. People not knowing the chosen parameter values used those octad class numbers in computer programs based on the Lamb-Zwolinski method. The best-fitting sets of parameter values obtained corresponded exactly to those initially set. The occurrence of multiple fits has been demonstrated and discussed; when multiple fits occurred, the parameter values in different ‘best fits’ were fairly similar in absolute values and very similar in relative values.

The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis.

Genetic recombination in meiosis plays an important role in generating diversity of genetic information. In yeast an Escherichia coli RecA-like gene, DMC1, is expressed in meiotic prophase and its product co-localizes with Rad51 protein on zygotene chromosomes. We have cloned the mouse and human homologs of the yeast DMC1 gene. The predicted human and mouse DMC1 proteins showed 54.1% sequence identity with yeast Dmc1 protein. The domain II region, highly conserved in the E.coli RecA-like protein family, was also found in the mammalian DMC1 proteins, including the two ATP binding motifs and DNA binding sites with the region. In situ hybridization analysis revealed expression of the mouse Dmc1 gene in testicular germ cells in meiosis; RT-PCR showed expression in embryonal ovaries. These findings suggest that DMC1 plays an important role in meiotic homologous recombination. From both the man and mouse we have isolated an alternative spliced form of Dmc1 cDNA (Dmc1-d), which is deleted for a region between the two motifs involved in nucleotide binding. Since the alternatively spliced Dmc1-d transcript was detected in both male and female germ cells, the encoded protein DMC1-D may have a novel role in mammalian genetic recombination in meiosis.

A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity.

Homologous recombination hot spots are DNA sites that increase the frequency of recombination in their vicinity. The M26 allele of the ade6 gene in Schizosaccharomyces pombe is the first meiotic hot spot with an identified unique nucleotide sequence. We have purified 40,000-fold a heteromeric protein, containing polypeptides Mts1 (70 kD) and Mts2 (28 kD), that binds to the M26 site. Binding in vitro strictly correlates with hot spot activity in vivo for numerous single base pair substitutions in the vicinity of the M26 site, indicating that Mts1/Mts2 activates the M26 site and promotes a rate-limiting step of meiotic recombination. These and other data suggest that homologous recombination may be regulated primarily by discrete DNA sites and proteins that interact with those sites.

Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a large number of genes in the RAD52 epistasis group has been implicated in the repair of chromosomal double-strand breaks and in both mitotic and meiotic homologous recombination. While most of these genes are essential for yeast mating-type (MAT) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion process. Using a galactose-inducible HO endonuclease gene to initiate MAT switching, we have examined the effect of null mutations of RAD50 and of XRS2 on intermediate steps of this recombination event. Both rad50 and xrs2 mutants exhibit a marked delay in the completion of switching. Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand break. The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells. However, later events are still further delayed, suggesting that XRS2 and RAD50 affect more than one step in the process. In the rad50 xrs2 double mutant, the completion of MAT switching is delayed more than in either single mutant, without reducing the overall efficiency of the process. The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad50 protein or to any other protein in the database. Overexpression of RAD50 does not complement the defects in xrs2 or vice versa.

Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a large number of genes in the RAD52 epistasis group has been implicated in the repair of chromosomal double-strand breaks and in both mitotic and meiotic homologous recombination. While most of these genes are essential for yeast mating-type (MAT) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion process. Using a galactose-inducible HO endonuclease gene to initiate MAT switching, we have examined the effect of null mutations of RAD50 and of XRS2 on intermediate steps of this recombination event. Both rad50 and xrs2 mutants exhibit a marked delay in the completion of switching. Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand break. The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells. However, later events are still further delayed, suggesting that XRS2 and RAD50 affect more than one step in the process. In the rad50 xrs2 double mutant, the completion of MAT switching is delayed more than in either single mutant, without reducing the overall efficiency of the process. The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad50 protein or to any other protein in the database. Overexpression of RAD50 does not complement the defects in xrs2 or vice versa.

RNA associated with a heterodimeric protein that activates a meiotic homologous recombination hot spot: RL/RT/PCR strategy for cloning any unknown RNA or DNA.

The ade6-M26 mutation in the fission yeast Schizosaccharomyces pombe creates a meiotic homologous recombination hot spot. We have achieved 40,000-fold purification of a heterodimeric DNA-binding protein, Mts1/Mts2, that activates the recombination hot spot. Physical studies suggested the presence of a third subunit. It is demonstrated here that RNA molecules of approximately 210 nucleotides copurified with the heterodimer. To characterize the RNA component, it was necessary to develop a new strategy for cloning of the unknown, low-abundance, partially degraded RNAs that were present in purified Mts1/Mts2 protein preparations. The strategy uses RNA ligase to add DNA oligonucleotide priming sites to the RNA for subsequent reverse transcription and PCR (RNA ligase, reverse transcription-PCR, or RL/RT/PCR). This cloning procedure could be applied to the cloning of any unknown RNA or DNA molecules. Because the cDNA clones obtained from Mts1/Mts2 were largely heterogeneous, it seems likely that the RNAs copurified as a result of tight but nonspecific interactions with the heterodimeric protein.

A DNA exonuclease induced during meiosis of Schizosaccharomyces pombe.

In meiotic cells of the fission yeast Schizosaccharomyces pombe, a DNA exonuclease activity increased approximately 5-fold after premeiotic S-phase and decreased to the initial level before the meiotic divisions. We have purified this activity, designated exonuclease I, to near homogeneity. The activity co-purified with a polypeptide with an apparent molecular weight of 36,000. With a linear double-stranded DNA substrate, exonuclease I degraded only the 5'-ended strand from each end to produce 3'-single-stranded tails. The enzyme also acted on nicked circular DNA with comparable affinity. The meiotic induction of exonuclease I and its mode of action, similar to that of recombination-promoting exonucleases from bacteria, suggest that exonuclease I is involved in meiotic homologous recombination in S. pombe.

Chromosomal context dependence of a eukaryotic recombinational hot spot.

The single base-pair mutation M26 in the ade6 gene of the fission yeast Schizosaccharomyces pombe creates a hot spot for meiotic homologous recombination. When DNA fragments containing M26 and up to 3.0 kilobases of surrounding DNA were moved to the ura4 gene or to a multicopy plasmid, M26 had no detectable hot spot activity. Our results indicate that nucleotide sequences at least 1 kilobase away from M26 are required for M26 hot spot activity and suggest that, as for transcriptional promoters, a second site or proper chromatin structure is required for activation of this eukaryotic recombinational hot spot. We discuss the implications of these results for studies of other meiotic recombinational hot spots and for gene targeting.