Checkpoint and DNA-repair proteins are associated with the cores of mammalian meiotic chromosomes

Checkpoint and DNA-repair proteins are associated with the cores of mammalian meiotic chromosomes

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4 Checkpoint and DNA-Repair Proteins Are Associated with the Cores of Mammalian Meiotic Chromosomes Madalena Tarsounas ∗ and Peter B. Moens Department of Biology York University Toronto, Ontario, M3J 1P3 Canada I. Introduction II. Structural Characteristics of Meiotic Chromosomes during the Prophase of Meiosis I A. Core Formation B. Meiotic Chromosome Synapsis C. SC Dissolution III. Meiotic Checkpoint and Recombination Proteins Are Associated with the Cores of the Meiotic Chromosomes A. The HR Model B. Recombination Proteins C. Checkpoint Proteins IV. Conclusions and Perspectives References

Meiotic checkpoints are manifested through protein complexes capable of detecting an abnormality in chromosome metabolism and signaling it to effector molecules that subsequently delay or arrest the progression of meiosis. Some checkpoints act during the first meiotic prophase to monitor the repair of chromosomal DSBs, predominantly by meiotic recombination, or to ensure the correct establishment of synapsis and its well-timed dissolution. In mammals, a number of checkpoint and repair proteins localize to the meiotic chromosomal cores, sometimes in the context of the synaptonemal complex (SC).1 Here we discuss possible functions of these proteins in the accomplishment of meiotic recombination and normal progression of the meiotic pathway. Also, we present arguments for a structural role of cores and SCs in the assembly of the repair and checkpoint protein complexes on the chromosomes.  2001 Academic Press. C

∗ Present address: Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, England. 1 Abbreviations used in the text: dsDNA, double stranded DNA; DSB, double stranded break; EM, electron microscopy; HR, homologous recombination; IR, ionizing radiation; LE, lateral element; NHEJ, nonhomologous end joining; SC, synaptonemal complex; SMC, structural maintenance of the chromosomes; ssDNA, single-stranded DNA; TF, transverse filament. Current Topics in Developmental Biology, Vol. 51 C 2001 by Academic Press. All rights of reproduction in any form reserved. Copyright  0070-2153/01 $35.00

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I. Introduction Meiosis is a special type of cell division in which two rounds of chromosome segregation follow one round of chromosome replication. Premeiotic DNA replication creates two copies of each parental chromosome (Fig. 1, see color insert). Each copy, termed homologous chromosome or homolog, consists of a pair of sister chromatids. During the prophase of meiosis I, connections are established between homologs and the resulting structure is referred to as a bivalent. At this stage, the synaptonemal complex, an assembly of nucleic acids and proteins positioned along the length of the homologs, brings the maternal and paternal chromosomes together, facilitating their recombination. The process of meiotic recombination accounts for the genetic variability in the germline and the phenotypic diversity of the progeny. The first meiotic division is reductional, that is, the two chromosomes in a bivalent, each consisting of a pair of sister chromatids, migrate to opposite poles. Sister chromatid and sister kinetochore cohesion are essential at this stage. At meiosis II (the equational division) the two sister chromatids in each chromosome separate and segregate to opposite poles. Overall, four haploid cells are generated from one diploid parental cell. When they become capable of undergoing fertilization, these haploid cells are identified as gametes. During sexual reproduction, fusion of two gametes of opposite mating types restores diploidy. The changes in chromosome morphology accompanying the progression through the meiotic pathway are usually referred to as the meiotic chromosome metabolism. Among them, recombination of meiotic chromosomes has a unique evolutionary significance, as it creates novel gene combinations in the progeny. Through natural selection, the genes that translate into successful survival and reproduction traits tend to spread within populations, ultimately leading to the appearance of new species. Therefore, meiosis can be viewed as the process that fueled evolution. In this context, it is obvious that evolutionary pressure for an error-free meiotic chromosome metabolism must have led to the development of rigorous surveillance mechanisms that monitor the correct accomplishment of each step of meiosis. Homologous recombination (HR) of the meiotic chromosomes is initiated during early meiotic prophase I with the formation of double stranded breaks (DSBs) in the chromosomes, which are repaired synchronously with the exchange of genetic information between homologous chromosomes. Elaborate activities for the repair of DSBs by HR have evolved, along with stringent checkpoint mechanisms that monitor repair and suppress errors. In most species, the role of these mechanisms is mainly to delay the transition from prophase I to subsequent meiotic stages (in mammals, the prophase I is the lengthiest stage of meiosis), in order to allow correct processing of the HR intermediates into the final recombination products. If an error exists, the checkpoints can arrest the progression of meiosis at the stage where the defect is detected.

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Many of the meiotic recombination and checkpoint activities are accomplished by proteins originating in somatic cells, from which they have probably been recruited during evolution into the meiotic process. Supporting this view is the fact that mutations in these genes cause genomic instability, massive cell death, and cancer due to DSB repair failure, and frequently, sterility due to impaired repair of meiotic DSBs. We summarize here some of the recent research on the structure of meiotic chromosomes during prophase I, as well as on the meiotic chromosome-associated proteins participating in the repair meiotic DSBs, or in the surveillance of meiotic progression. The focus of this review is on the structure of mammalian meiotic chromosomes and on the proteins associated with them that play repair and surveillance roles in the maintenance of genomic integrity.

II. Structural Characteristics of Meiotic Chromosomes during the Prophase of Meiosis I Following DNA replication, a genome-wide search for homology occurs and, as a result, homologous chromosomes align with each other side-by-side, a configuration that is stabilized by transient paranemic DNA–DNA interactions (Weiner and Kleckner, 1994). This process is termed homolog pairing and it precedes synapsis, the stage at which the homologs are intimately associated in the context of the SC. In most organisms, pairing is initiated at leptotene, the first structurally defined stage of prophase I (Fig. 2, see color insert), but despite intensive investigation, very little molecular detail has been accumulated on this process (for reviews, see Roeder, 1997; Zickler and Kleckner, 1998). The presence of certain proteins such as Saccharomyces cerevisiae Hop2 has been shown to be required for homolog pairing: In its absence, normal levels of synapsis are detected, but this is entirely established between nonhomologous chromosomes (Leu et al., 1998). This phenotype indicates that ectopic DNA interactions may occur during prophase I, and specialized proteins are required to prevent pairing and synapsis between nonhomologous chromosomes. In mammals, such a role may be played by the recombination protein DMC1, as the spermatocytes of Dmc1−/− mice sometimes exhibit nonhomologous synapsis (Yoshida et al., 1998).

A. Core Formation Simultaneously with chromosome pairing, proteinaceous cores (also termed axial elements or chromosome axes) form along each homolog. In rodents and probably other eukaryotes, the cores represent anchorage sites for the chromatin loops (Heng

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et al., 1994, 1996; Moens et al., 1998). Sequencing of the DNA present in the rodent SCs has demonstrated an enrichment in LINE and SINE fragments (Pearlman et al., 1992). Biochemical evidence for the DNA-binding role of the cores was obtained from recent studies in yeast indicating the ability of the core component Hop1 (see below) to bind duplex DNA in vivo and in vitro (Kironmai et al., 1998). At synapsis, the cores form the lateral elements (LEs) of the SC (Fig. 2). It is postulated that they may represent the structures to which the transverse filaments (TFs) attach in order to effect synapsis. Two protein components of the cores are known in the yeast S. cerevisiae: Hop1 and Red1 (Hollingsworth and Byers, 1989; Rockmill and Roeder, 1990). They colocalize on the prophase I chromosomes, where they are found in the unsynapsed cores and mature SCs. Red1 and Hop1 dissociate from the chromosomes as the SC disassembles (Smith and Roeder, 1997). Red1 is required for LE assembly (Rockmill and Roeder, 1990), as well as for the assembly of Hop1 on the chromosomes (Smith and Roeder, 1997). Therefore it is postulated that Red1 nucleates the formation of axial elements in yeast (Roeder, 1997). red1 and hop1 mutants lack SCs and exhibit residual levels of recombination and chromosome nondisjunction, which causes severe spore inviability (Hollingsworth and Byers, 1989; Rockmill and Roeder, 1990; Mao-Draayer et al., 1996). This points to a major role for the correct core formation in recombination and the successful completion of meiosis. Remarkably, the core components in budding yeast have no similarity at the amino acid level with potential functional homologs from other eukaryotes. This conclusion has been based initially on the observed lack of cross-reactivity of polyclonal antibodies between eukaryotic species (S. cerevisiae, Drosophila, Xenopus; P. B. Moens, unpublished) and was subsequently confirmed by the identification of core protein components in a number of different species and comparison of their amino acid sequences. Determining the protein composition of the cores in rodents has represented a challenge for quite some time because of difficulties in the isolation of these chromosome-associated proteins. This work was pioneered by Heyting et al. (1985) with the isolation of SCs from rat spermatocytes. The SCs have been injected in mice and rabbits, and the antibodies generated recognized the cores and the SC when tested by indirect immunofluorescence (Moens et al., 1987; Heyting et al., 1988; Moens et al., 1992). In immunological screenings of expression libraries from rodent testis, the cDNAs encoding the core components COR1(SCP3)2 (Dobson et al., 1994; Lammers et al., 1994) and SCP2 (Offenberg et al., 1998), as well as the synaptic protein SYN1(SCP1) (Dobson et al., 1994), were isolated. SCP2 was also isolated in a two-hybrid screen of a testis cDNA 2 This protein component of the cores is termed COR1 in hamster and SCP3 in rat. Similarly, the synaptic protein is termed SYN1 in hamster and SCP1 in rat. Here we use a compound name that includes both denominations for each protein.

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library with COR1(SCP3) as a bait (Tarsounas et al., 1999b). These three proteins are expressed exclusively during meiosis. The distribution of COR1(SCP3) in the chromosome cores and LEs (Fig. 2) was studied with specific antibodies raised against the full-length protein (Dobson et al., 1994; Lammers et al., 1994). COR1(SCP3), a Mr 30,000/33,000 protein, appears as short segments on the chromosomes at leptotene. The staining becomes continuous at zygotene when it clearly marks the cores in the process of alignment. At pachytene, the two homologous cores are in close juxtaposition and the COR1(SCP3) staining identifies bivalents, rather than individual homologs at the light microscopy level of resolution. At diplotene, anti-COR1(SCP3) antibodies stain the separating cores and clearly mark chiasmatic configurations. Consistent with an essential role for COR1(SCP3) in forming the cores of the meiotic chromosomes, the Cor1(Scp3)−/− mice lack the potential to assemble fully developed SCs (Yuan et al., 2000). The continuous presence of COR1(SCP3) along the chromosomes at all prophase I stages and its disappearance at the first meiotic division indicate a possible role in sister chromatid cohesion. In addition, COR1(SCP3) is detected at anaphase II in the space between the separating kinetochores (Moens and Spyropoulos, 1995). Its persistence at the kinetochores until anaphase II suggests a possible role in establishing and maintaining sister kinetochore cohesion until sister chromatid separation, a role similar to that of Drosophila Mei-S332 (Kerrebrock et al., 1995; Moore et al., 1998) and Schizosaccharomyces pombe cohesin comples (Tanaka et al., 1999). COR1(SCP3) participates in homotypic interactions in a two-hybrid system and in vitro that may be mediated by coiled-coil formation (Tarsounas et al., 1997). This observation suggested that COR1(SCP3) may assemble multimeric aggregates on the chromosomes. This seems indeed to be the case, as Yuan et al. (1998) have shown that the mouse homolog of COR1(SCP3), termed SCP3, assembles multistranded fibers in the nucleus and cytoplasm of fibroblasts in culture. A region with coiled-coil forming potential is required for the assembly of COR1(SCP3) fibers, which confirmed the importance of this region for the COR1(SCP3) homotypic interactions (Tarsounas et al., 1997). These fibers are structurally related to the intermediate filaments; hence the conclusion that COR1(SCP3) assembly occurs with the formation of filamentous structures along the chromosomes. COR1(SCP3) assembly most likely does not require the presence of DNA because COR1(SCP3) filaments can form in the cytoplasm of the transfected cells (Yuan et al., 1998) and cytoplasmic poly-SC complexes are detected in several organisms (Goldstein, 1987). However, it does not exclude the possibility that these filaments have DNA binding ability, as the chromatin loops appear embedded in the chromosomal cores at the level of resolution of immunofluorescence microscopy (Heng et al., 1997). Studies of chromatin organization in the Cor1(Scp3)−/− mice are currently in progress.

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A second component of the chromosome cores in rat spermatocytes has been characterized (Offenberg et al., 1998). This large protein, termed SCP2 (Mr 190,000), has a distribution pattern on the chromosomes similar to COR1(SCP3) (Offenberg et al., 1998; Schalk et al., 1998). The C-terminal fragment of the hamster homolog of the SCP2 protein has also been isolated in a two-hybrid screen of a hamster cDNA library using COR1(SCP3) as a bait (Tarsounas et al., 1999b). This fragment possesses coiled-coil forming ability, which demonstrates that this motif is important for the interaction between the COR1(SCP3) and SCP2 proteins as well as for homotypic COR1(SCP3) interactions. It is possible therefore that the two proteins form a structural complex of the meiotic chromosome cores. SCP2 and COR1(SCP3) may form individual filaments that assemble into a higher order structure. Alternatively, they may form mixed filaments in which SCP2 and COR1(SCP3) monomers assemble into the cores lining the chromosomes, possibly with a defined stoichiometry of the two monomers. The second scenario seems more probable, in the light of the observation that Cor1(Scp3)−/− mice do not form axial elements (Yuan et al., 2000). Studying the distribution of the SCP2 protein in these knockout mice will bring further insight into this matter. Similarly to COR1(SCP3) and SCP2 proteins, the yeast proteins Red1 and Hop1, which are both structural components of the yeast chromosome cores (Hollingsworth and Byers, 1989; Rockmill and Roeder, 1990; Mao-Draayer et al., 1996), interact with themselves and with each other in the axial elements (Hollingsworth and Ponte, 1997). Moreover, overexpression of Red1 has a negative effect on spore viability because a strict Red1/Hop1 stoichiometry is required for the axial element assembly and normal chromosome segregation at meiosis (Friedman et al., 1994). In the model proposed by Hollingsworth and Ponte (1997), the chromosomal cores assemble from Red1 and Hop1 monomers, and phosphorylation by Mek1 kinase is required for maintaining the structural stoichiometry. We entertain the possibility that although individual components do not share sequence similarity, the overall structure of the cores/LEs is evolutionarily conserved from yeast to mammals. A new dimension in the structure of meiotic chromosomes has been introduced by the discovery of structural maintenance of chromosomes (SMC) proteins in association with meiotic chromosomes. SMCs represent a family of heterodimeric proteins with established mitotic roles in chromosome condensation and gene dosage compensation (SMC2 and SMC4), and sister chromatid cohesion (SMC1 and SMC3; reviewed by Strunnikov, 1998; Hirano, 1999). In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the protein complex required for sister chromatid cohesion during mitosis, termed cohesin, is also functional at meiosis, where it plays a role in sister chromatid cohesion, core formation, and recombination, but at least one of its components, Rec8 is meiosis-specific (reviewed by Orr-Weaver, 1999). Eijpe et al. (2000) have found that the heterodimer SMC1/SMC3 is associated with the cores/SCs of the rat meiotic chromosomes, supporting the role previously

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proposed for cores/SCs in sister chromatid cohesion throughout prophase I of meiosis (Bickel and Orr-Weaver, 1996). The direct interactions determined between SMC1/SMC3 and core/SC components SCP1 and SCP2 (Eijpe et al., 2000) suggest that SMCs are required for the core assembly. Also, mammalian SMCs may bind chromatin directly and anchor it to the axial element of the meiotic chromosomes, similarly to their proposed mitotic function (Hirano, 1999). A role for mammalian SMCs in meiotic recombination is expected by analogy with yeast (Eijpe et al., 2000).

B. Meiotic Chromosome Synapsis Synapsis is accomplished when the axial elements delineating each homolog come in close proximity and become connected by the transverse filaments located in the central region of the SC (Fig. 2). The SYN1(SCP1) protein in rodents, also called the synaptic protein, has a critical role in establishment of chromosome synapsis (Meuwissen et al., 1992; Dobson et al., 1994; Schmekel et al., 1996). SYN1(SCP1) is thought to be the molecular component of the TFs that closes as a zipper at synapsis (Fig. 2). SC assembly initiates at zygotene, synchronously with the nuclear appearance of SYN1(SCP1) in regions where the homologs are intimately connected. These regions, which are positioned randomly along the length of rodent chromosomes, will extend to form fully synapsed chromosomes at the pachytene stage (Meuwissen et al., 1992; Dobson et al., 1994). Epitope mapping of the Mr 125,000 SYN1(SCP1) with electron microscopy (EM) revealed that its C terminus is located in the LE and the N terminus in the center of the SC (Fig. 2; Dobson et al., 1994; Schmekel et al., 1996; Liu et al., 1996). A similar configuration has been demonstrated for Zip1, a possible functional homolog of SYN1(SCP1) in yeast (Tung and Roeder, 1998). This supports the assumption that the TFs, cytologically positioned in the central region and perpendicular to the LE, may be formed by individual SYN1(SCP1) or Zip1 molecules. The N-terminal 100 amino acids of the SYN1(SCP1) protein, which lack the potential of forming coiled-coil motifs, self-interact in a two-hybrid system (Liu et al., 1996). Regions mapping to the middle and C-terminus of the SYN1(SCP1) molecule with high probability of forming coiled-coils do not participate in homotypic interactions (Tarsounas et al., 1997). This distribution supports the idea that SYN1(SCP1) forms the TFs in the central region of the SC, positioned perpendicularly to the LEs and connected in the centre of the SC by direct interactions between the N termini of molecules positioned opposite each other (Fig. 2). Detection of such interactions in a two-hybrid system, however, does not exclude the possibility that other proteins may act as adapters between the N-terminal regions of SYN1(SCP1) in the central region of the SC. A two-hybrid interaction was detected between full-length mouse SYN1(SCP1) molecules, but not between full-length SYN1(SCP1) and COR1(SCP3) (Tarsounas

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et al., 1997), which suggests that most likely COR1(SCP3) is unlikely to be required for the anchorage of SYN1(SCP1)-formed TFs to the lateral regions of the SC. As Cor1(Scp3)−/− spermatocytes are incapable of assembling SYN1(SCP1) and the central region correctly (Yuan et al., 2000), it is possible that the severe general defect in core formation in these cells also affect SYN1(SCP1) positioning on the chromosomes. An as-yet unknown component of the SC may act as an adapter between the TFs and the lateral region of the SC, and its localization may be dependent upon COR1(SCP3) filament formation. In budding yeast, the Zip1 protein has been shown to be a central region component (Sym et al., 1993, 1995) with similar meiotic functions as SYN1(SCP1). In addition, a second protein termed Zip2 is required for establishment of synapsis in yeast. Analysis of Zip1 and Zip2 distributions in the zip2 and zip1 mutants, respectively, suggest that Zip2 plays a structural role, being required for the assembly of Zip1 on the chromosomes (Chua and Roeder, 1998). Zip2 has been shown to localize to the “axial associations,” which are bridgelike structures connecting the LE at a few sites along their length before synapsis, and may also represent recombinogenic sites as suggested by the presence of Rad50 protein (Chua and Roeder, 1998). This finding supports the assumption that recombination intermediates are required for initiation of synapsis. Consistent with this, a spo11 mutant with a DSB formation defect does not assemble SCs (Cha et al., 2000). In mammals, only a limited number of SC components have been identified thus far, and no protein sharing the structural features of Zip2 is yet known. The direct interaction detected between RAD51 and SYN1(SCP1), as well as the presence of the RAD51/DMC1 complexes between the homologous axes before synapsis (Tarsounas et al., 1999b), suggest that RAD51 may recruit SYN1(SCP1) at the synapsis initiation sites, from where synapsis subsequently extends along the chromosomes.

C. SC Dissolution SC disassembly starts during late pachytene with the gradual removal of SYN1(SCP1) from the separating chromosome cores (Tarsounas et al., 1999a). At early diplotene, only short stretches of SYN1(SCP1) remain visible along the chromosomes, and they disappear by late diplotene (Moens and Spyropoulos, 1995). The COR1(SCP3) protein remains in the axes of the post-prophase I chromosomes until the first meiotic division, and it is lost thereafter from this location. COR1(SCP3) is last detected at the separating anaphase II kinetochores (Moens and Spyropoulos, 1995). The nature of the signal that triggers the dissolution of the SC has not yet been established. It is very likely that the molecules that monitor meiotic progression will transmit a signal to the structural blocks that form the SCs once meiotic recombination has been successfully completed. This may cause SC components to

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gradually dissociate from the chromosomes and probably be targeted for degradation thereafter. One possibility is that phosphorylation triggers this series of events. Several potential protein kinase target sites have been revealed by analysis of the amino acid sequences of the three mammalian SC components COR1(SCP3), SYN1(SCP1), and SCP2. Direct evidence that the SC components COR1(SCP3) and SYN1(SCP1) are phosphoproteins in pachytene spermatocyte extracts has been reported by Lammers et al. (1995) and Tarsounas et al. (1999a). Alternative mechanisms such as ubiquitination (Tarsounas et al., 1997) should also be investigated.

III. Meiotic Checkpoint and Recombination Proteins Are Associated with the Cores of the Meiotic Chromosomes A concrete structural function for the SC during meiosis has not yet been established in mammalian cells. One hypothesis addressed in a review by Moens et al. (1998) is that the cores represent attachment sites for the chromatin loops, thus contributing to the general organization of the meiotic chromosomes. Following premeiotic replication, the bases of the loops of the two sister chromatids are attached to the protein cores (Fig. 2). The central SC region, which assembles later in meiosis, then establishes the physical connection between the homologs, possibly creating a steric environment favorable for recombination events to take place (reviewed in Roeder, 1997). Increasing evidence supports a novel role of the cores/SCs in HR by providing the structural matrix for the assembly of DSB repair complexes. Meiotic recombination involves the formation of DSBs along the length of the chromosomes and their repair with concomitant exchange of genetic information between the maternal and paternal chromosomes. Much of what we currently know about meiotic HR actually comes from studies in the budding yeast, S. cerevisiae (reviewed by Smith and Nicolas, 1998). Yeast is a very popular model, mainly because it offers the facility to induce meiosis (upon removal of essential nutrients) and to obtain synchronous populations of cells at various meiotic stages in which HR can be monitored both biochemically and cytologically (Padmore et al., 1991). Generation of single and multiple mutants in HR genes helps in gaining insight into their function (Kleckner, 1996; Roeder, 1997). In yeast, chromosome synapsis was assessed in recombination- or sporulation-deficient mutants, and vice versa, and a correlation has been established between the two. In most eukaryotes, chromosome synapsis is required for normal levels of recombination. Conversely, SC formation may require at least some of the recombination steps to be correctly completed (Bishop, 1994; Rockmill et al., 1995; Nairz and Klein, 1997). The fission yeast S. pombe represents an exception to this rule because it lacks SCs, but exhibits normal levels of meiotic recombination. However, structures resembling the cores have been detected, and these may be sufficient for the meiotic recombination to

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occur (Scherthan et al., 1994). The lack of synapsis correlates with the absence of crossover interference in this organism (Kohli and Bahler, 1994). Similar functional studies in mammals involve generation of knockout mice and are very laborious. In addition, some knockouts are embryonic lethal (e.g., Rad51−/−); therefore, a meiotic phenotype is not available. Indirect evidence comes from biochemical studies of mammalian enzymatic activities in vitro, protein–protein interactions, and the patterns of immunolocalization of these proteins on the meiotic chromosomes. Recent data suggest that proteins directly involved in recombination and in monitoring repair of meiotic DSBs by HR are associated with the meiotic chromosomes. These are primarily proteins involved in DNA damage repair and damage-induced checkpoint control during the cell cycle. These proteins are presumably recruited to perform similar repair and surveillance functions during meiosis. It is particularly interesting that their association with meiotic chromosomes does not occur randomly: These proteins are specifically positioned on the cores of meiotic chromosomes, where the chromatin loops also attach. This led to the speculation that crucial meiotic events such as HR occur within the context of the SC, between DNA sequences located at the base of the chromatin loops. The recently reported phenotype of Cor1(Scp3)−/− mice supports this hypothesis. The absence of COR1(SCP3) changes the nuclear distribution of repair and recombination proteins such RAD51 and RP-A (Yuan et al., 2000), suggesting a structural role for the axial elements/SCs in HR. We present here the immunolocalization patterns on the mammalian meiotic chromosomes reported for some recombination and checkpoint proteins, and the functional aspects inferred from them. It is worth mentioning that although the data summarized here have been obtained exclusively from examination of spermatocyte nuclei, it is generally assumed that similar events governed by similar proteins occur during female meiosis.

A. The HR Model The working model for the succession of recombination events in eukaryotes predicts the formation of DSBs on one of the homologous chromosomes (Szostak et al., 1983; Fig. 3, see color insert). In experiments using the well-characterized HIS4-LEU2 DSB “hot-spot” (Cao et al., 1990) in S. cerevisiae, Padmore et al. (1991) showed the correlation between chromosome synapsis and the succession of recombination events leading to the repair of meiotic DSBs. Evidence for the generation of meiotic DSBs in organisms other than yeast is currently lacking. Despite this limitation, it is assumed that the meiotic recombination model based on DSBs is valid among all eukaryotes. DSBs appear prior to or concomitant with the initiation of axial core assembly on the chromosomes at the leptotene stage of prophase I, and they persist until pachytene when the SC is fully assembled on the chromosomes. DSBs represent

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substrates for an exonuclease activity that resects the DSBs leaving 3 singlestranded DNA (ssDNA) tails. These ssDNA tails, which represent binding sites for RecA homologs, may have the ability to assess sequence identity or similarity in another DNA molecule and to invade an intact DNA duplex on a homologous chromosome, leading to the formation of a Holliday junction. This may coincide with initiation of chromosome synapsis. Following branch migration, this intermediate structure is resolved by cleavage and ligation activities, resulting in a crossover or noncrossover product. These recombinant products are cytologically visible at the end of the pachytene stage when desynapsis initiates, as chiasmata, sites of constriction along the length of the chromosomes.

B. Recombination Proteins 1. SPO11 The catalytic subunit of the meiosis-specific enzymatic activity that produces DSBs was originally identified in yeast as Spo11, a member of a conserved topoisomerase family represented in fission yeast, nematodes, and archaebacteria (Keeney et al., 1997; Bergerat et al., 1997; Dernburg et al., 1998). Identification of Spo11 as the meiotic DNA cleavage activity strongly suggests that meiotic DSBs occur by a topoisomerase-like transesterification reaction, rather than by endonucleolytic hydrolysis. This observation has important consequences for the meiotic progression, a major one being that, since topoisomerase-mediated breaks can be reversed, it is possible to reverse meiotic DSBs in the absence of a suitable recombination partner (Keeney et al., 1997). This reversibility may also explain why only a small fraction of meiotic DSBs lead to successful crossover events. The human and mouse homologs of Spo11 have been identified and found to be transcribed and expressed from the early stages of meiosis (leptotene/zygotene) to mid-pachytene (Keeney et al., 1999; Shannon et al., 1999; Romanienko and Camerini-Otero, 1999), which suggests a role for this protein during the late stages of prophase I as well. Antibodies against mammalian SPO11 preferentially stain the SCs along their length from mid- to late pachytene (P. Baudat and S. Keeney, personal communication). This pattern coincides with the one reported for mammalian topoisomerase II by Moens and Earnshaw (1989). The presence of topoisomerases on the SC at this stage indicate either that DSBs are made in higher eukaryotes at a later stage than in yeast, that is, after synapsis is completed (Shannon et al., 1999), or that topoisomerases play a structural as well as enzymatic role during meiosis. 2. MRE11/RAD50/NSB1 These human proteins have been identified based on their structural (MRE11/ RAD50) and functional (NBS1) homology to the well-characterized yeast Mre11/

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Rad50/Xrs2 repair complex. The three proteins interact with each other forming IR-induced DNA repair complexes, visualized as immunofluorescent foci in the nuclei of somatic cells (Maser et al., 1997). The enzymatic activity of these complexes is mediated by MRE11 (reviewed by Haber, 1998), a nuclease that is essential for genomic stability and cell viability in human cells (Yamaguchi-Iwai et al., 1999). While RAD50 enhances the enzymatic activity of MRE11 (Paull and Gellert, 1998), no concrete function has been assigned to NBS1, except for a putative structural requirement in the assembly of RAD50/MRE11 at sites of DNA damage (Carney et al., 1998). The NBS1 gene has been shown to be mutated in the Nijmegen breakage syndrome (NBS) (Carney et al., 1998; Varon et al., 1998), a chromosomal-instability syndrome whose symptoms include increased cancer incidence, cell cycle checkpoint defects, and ionizing radiation sensitivity; this is the first implication of this protein in a DNA repair pathway. The MRE11/RAD50/NBS1 complex is required for the repair of DSBs by both the HR and the NHEJ pathway in both meiotic and mitotic cells (reviewed by Haber, 1998; Dasika et al., 1999). The nuclease activity of this complex creates 3 overhangs that may function as binding sites for the proteins involved in strandexchange reactions (Fig. 3). The yeast complex has been proposed to be also active in remodeling the chromatin into a structure that facilitates DSB formation (Usui et al., 1998). At meiosis, mammalian RAD50 and MRE11 show identical temporal and spatial localization patterns, with abundant diffuse nuclear staining at leptotene/zygotene (Goedecke et al., 1999). At this stage both DSB formation and resection are assumed to occur, confirming the assigned role in DSB processing. The fact that the MRE11/RAD50 complexes are not specifically positioned on the meiotic chromosome cores is consistent with the notion that at this stage DSBs occur randomly throughout the nuclear chromatin. The persistence of these complexes to mid-pachytene in association with specialized spermatocyte domains, such as the sex vesicle (Goedecke et al., 1999), has an unknown biological significance. 3. RAD51 and DMC1 These mammalian RecA homologs share a high degree of identity at the amino acid level and exhibit similar catalytic activities in vitro, including the ability to promote homologous DNA pairing and strand transfer reactions (Baumann et al., 1996; Baumann and West, 1997; Li et al., 1997; Masson et al., 1999). RAD51 is expressed in both somatic and meiotic tissues and its deficiency is lethal early in embryogenesis (Lim and Hasty, 1996; Tsuzuki et al., 1996), suggesting an essential function for cell viability. In somatic cells in culture, it is postulated that repair complexes are assembled at the sites of DSBs upon DNA-damage induction, although methods for direct visualization of the DSBs in these cells are not currently available. RAD51 is an essential component of the initial repair complexes, and its multimers can be detected as nuclear foci by indirect immunofluorescence. Many other proteins involved in DNA repair by HR, such as RAD52, RP-A, RAD54,

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BRCA1, and BRCA2, are also recruited to these sites (Dasika et al., 1999). The Dmc1 gene is specifically expressed in meiosis, and its null-mutation in mice causes meiotic arrest at the zygotene stage without homolog synapsis (Pittman et al., 1998) or with occasional synapsis between nonhomologs (Yoshida et al., 1998). This characteristic phenotype substantiates the role of DMC1 in promoting interactions between homologous chromosomes, which appears to be conserved from yeast to mammals (Schwacha and Kleckner, 1997). During the early stages of meiosis, RAD51 and DMC1 proteins have been shown in various species to form complexes visualized by immunofluorescence microscopy as discrete foci along the chromosome cores (Bishop, 1994; Terasawa et al., 1995; Dresser et al., 1997; Anderson et al., 1997; Moens et al., 1997; Barlow et al., 1997a; Tarsounas et al., 1999b). Immunogold EM with antibodies that recognize specifically each protein showed that the two are always present in mixed complexes on the chromosomes. These complexes are most likely formed by direct protein–protein interactions between the two recombinases (and possibly other proteins), as shown in vitro and in a two-hybrid system (Tarsounas et al., 1999b; Masson et al., 1999). Because of their ability to form multimeric filaments on ssDNA in vitro, the RAD51/DMC1 complexes have been proposed to coincide with the sites of resected DSBs (Bishop, 1994). Supporting this assumption is the decrease in the numbers of RAD51/DMC1 foci from leptotene/zygotene to pachytene that correlates with the disappearance of DSB. Therefore, in the early steps of meiotic recombination RAD51 and DMC1 may act directly in the formation of joint molecules by strand-exchange reactions (Fig. 3), consistent with their potential demonstrated in vitro. 4. RP-A Replication protein A (RP-A) is a ssDNA binding protein involved in DNA replication, repair, and recombination in mitotic cells (reviewed by Wold, 1997). The biochemical properties of the heterotrimeric RP-A protein include the potential to enhance efficiency of strand transfer reactions promoted by RecA homologs (reviewed by Baumann and West, 1998). This synergistic action involves RP-A binding to the ssDNA and removal of secondary structures, thus allowing RAD51 to form continuous filaments. Consistent with these biochemical data, RP-A and RAD51 colocalize extensively at discrete foci in meiotic cells and mitotic cells exposed to genotoxic treatments (Fig. 4, see color insert; Plug et al., 1998; Golub et al., 1998). However, in rodent spermatocytes there is a significant number of sites at which the two proteins do not colocalize when visualized by immunofluorescence microscopy (Figs. 4A and 4B) and, more obviously, by immunogold labeling in EM preparations (Figs. 4C and 4D). The most striking example is perhaps the X chromosome of the XY pair in which RAD51/DMC1 complexes are abundant (Fig. 4D), but RP-A cannot be detected. Also, the pseudoautosomal region of the

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XY pair, which is postulated to contain an obligatory crossover and contains at least one site of RAD51/DMC1 accumulation, lacks RP-A staining (Fig. 4D). This differential positioning may be explained by the fact that, at the early pachytene stage, RP-A is abundant on the cores of the meiotic chromosomes and the SCs, practically coating them along their entire length (Fig. 4A). Consequently, RP-A probably competes with RAD51 for the potential ssDNA binding sites, and either displaces RAD51 or delays its assembly. The sites where the two proteins colocalize are probably the recombinogenic sites where RP-A enhances the ability of RAD51 to promote strand exchange, as predicted by the biochemical data. This example of RAD51 and RP-A immunolocalization on meiotic chromosome axes is a striking example of the enhanced resolution obtained when gold grains are used for antigen detection by EM, as opposed to the regular immunostaining techniques. 5. MLH1 A role for homologs of the Escherichia coli mismatch repair apparatus has been demonstrated in the late steps of meiotic recombination, primarily in the correction of mismatches in heteroduplex DNA (Stahl, 1996; Roeder, 1997). Three genes, mutS, mutL, and mutH, are essential for the correction of replication errors in E. coli: MutS recognizes the mismatch and MutH protein acts in a complex with MutS and MutL as an endonuclease that nicks the newly synthesized strand (for a review see Modrich, 1991). In humans, alterations in the products of the mutS homologs, msh2, msh4, and msh5, as well as the mutL homologs mlh1 and pms2 are associated with various forms of cancer (Baker et al., 1995, 1996). The idea that mutations in these mismatch repair genes could have consequences in the germline through perturbation of meiotic events such as genetic recombination and homolog synapsis, was confirmed by gene targeting experiments in mice. Occasional sterility (Mlh1) accompanied by pairing defects, fragmentation of the axial elements of the SC, and nonhomologous synapsis represent the most common abnormalities of the mice carrying null mutations in Pms2 (Baker et al., 1995), Mlh1 (Edelmann et al., 1996; Baker et al., 1996), and Msh5 (Edelmann et al., 1999). Probably the most likely candidate for a protein directly involved in the late steps of meiotic recombination is MLH1, which is the only mismatch repair protein that has been shown to localize on the mouse and human meiotic chromosomes at discrete foci representing late recombination nodules (Baker et al., 1996; Barlow and Hulten, 1998). The late recombination nodules are electron-dense structures, positioned along the SCs and thought to harbor the enzymatic activities required for the late steps of meiotic recombination (Roeder, 1997). These late recombination nodules evolve into chiasmata. In mouse spermatocytes, the MLH1 protein immunolocalizes to an average of 1.2 foci per SC at pachytene (Hassold, 1996), which corresponds to the expected number of chiasmata per bivalent. In addition,

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air-dried human nuclear preparations for light microscopy revealed that the total number of MLH1 foci per nucleus at the mid- to late pachytene stage is comparable to the total number of chiasmata per nucleus (Barlow and Hulten, 1998). The distribution pattern of MLH1 in mouse pachytene spermatocytes is dependent on the SC length and shows crossover interference (Anderson et al., 1999). These observations indicate that MLH1 may indeed mark the sites of crossing over on the meiotic chromosomes. Therefore, MLH1 is thought to promote HR and act synergistically with the recombination machinery of the cell, possibly by suppressing insertion/deletion mispairs introduced during Holliday junction processing. 6. BLM and WRN Whereas RAD51 and DMC1, the eukaryotic homologs of the RecA strand exchange protein of bacteria, have been thoroughly investigated, very little is known about the proteins involved in the late steps of HR, including branch migration and resolution. The BLM and WRN proteins are encoded by genes mutated in the Bloom and Werner syndrome, respectively, two genetic diseases that result in similar cellular and clinical phenotypes, including genomic instability and a high incidence of cancer (German, 1993; Yu et al., 1996). Both proteins have helicase domains and are able to complement the mutation of sgs1 helicase gene in S. cerevisiae (Yamagata et al., 1998), which makes them potential candidates for enzymes involved in the branch migration step of recombination. To date, the WRN protein has not been reported to be associated with the progression of meiosis. Antibodies against this protein do not stain any discernible structures on rodent meiotic chromosomes (P. B. Moens, unpublished data). In mammalian mitotic cells WRN is found in the nucleolus (Marciniak et al., 1998). Two reports (Walpita et al., 1999; Moens et al., 2000) have shown the BLM protein to play a role during meiosis. From the data accumulated thus far, it is not clear whether this role is in promoting or suppressing HR. On meiotic chromosomes, BLM is concentrated in foci that, at the level of resolution of EM, colocalize significantly with the RAD51/DMC1 foci at the zygotene/early pachytene stages of prophase I (Moens et al., 2000). This observation may suggest that BLM acts synergistically to RAD51/DMC1 during HR. In addition, mouse spermatocytes show an excess of BLM protein in the pseudoautosomal region of the XY pair that is highly recombinogenic. Arguments for the role of BLM in suppressing, rather than promoting, HR are based on the hyperrecombination phenotype of BS cells (Watt et al., 1996), which suggests that BLM may prevent heteroduplex formation. The number of DSBs made during early prophase I exceeds the number of recombination products estimated from the number of chiasmata. Therefore, it is possible that helicases such as BLM will act at the noncrossover sites to reverse branch migration and suppress HR. These nonrecombinogenic DSBs can be repaired by alternative pathways,

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such as nonhomologous end joining (NHEJ; reviewed by Critchlow and Jackson, 1998). 7. BRCA1/BRCA2 BRCA1 and BRCA2 are primarily known because germline mutations in the corresponding genes confer a high risk of breast and ovarian cancer in the affected individuals (for a review, see Welcsh et al., 2000). BRCA1 and BRCA2 encode large nuclear proteins with no detectable amino acid sequence homology to each other. However, the two proteins may have synergistic functions in DNA damage repair pathways. Their involvement in DNA repair was primarily suggested by the high sensitivity of BRCA1−/− and BRCA2−/− cells to various DNA-damaging agents (Connor et al., 1997; Sharan et al., 1997; Gowen et al., 1998; Shen et al., 1998; Chen et al., 1998; Patel et al., 1998; Abbott et al., 1999). These cells also exhibit aneuploidy and other chromosomal aberrations (Marcus et al., 1996; Patel et al., 1998) indicative of a role of BRCA1 and BRCA2 in the maintenance of genomic stability and DSB repair (Moynahan et al., 1999). Furthermore, BRCA1 and BRCA2 colocalize with each other upon induction of DNA damage in mitotic cells (Chen et al., 1998) and interact directly with RAD51 (Scully et al., 1997; Sharan et al., 1997; Wong et al., 1997). Both proteins are expressed at high levels in meiotic cells (Zabludoff et al., 1996; Rajan et al., 1997) and colocalize on the unsynapsed cores of meiotic chromosomes during zygotene and on the SCs during pachytene (Chen et al., 1998). The mixed BRCA1/BRCA2 foci in general coincide with RAD51 complexes, suggesting that the three proteins act together in the repair of DSBs during meiotic recombination, but their precise mechanism of action is not known.

C. Checkpoint Proteins 1. ATM and ATR ATM and ATR belong to the PI3 -kinase-related (PIK) protein kinase superfamily (reviewed by Hoekstra, 1997) and are thought to participate in a meiotic and mitotic surveillance mechanism. Mutation in the ATM gene causes the human genetic disorder ataxia telangiectasia (A-T), characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DSBs persist in A-T cells after irradiation, because of impaired HR-mediated DSB repair (Morrison et al., 2000). The best-characterized substrate for the kinase activities of ATM and ATR in mitotic cells is p53 (Canman et al., 1998; Banin et al., 1998; Tibbetts et al., 1999; Lakin and Jackson, 1999), which can be phosphorylated by either kinase in response to different types of DNA damage (ionizing radiation or UV-induced damage, respectively). ATM interaction with dsDNA in vitro is

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enhanced by IR (Suzuki et al., 1999). Subsequently, a model has been proposed in which ATM is capable of detecting the DNA damage directly and signaling it to cell cycle suppressor proteins, such as p53. Other substrates for ATM include RP-A and NBS1, both involved in DSB repair (Dasika et al., 1999), as well as c-Abl, which most likely phosphorylates RAD51 in vivo, inducing its recruitment to DSBs upon IR-induced DNA damage (Chen et al., 1999). The functional significance of these phosphorylation reactions for the cellular response to DNA damage is not yet understood. Atm−/− mice have both mitotic and meiotic defects (Barlow et al., 1996; Xu et al., 1996). These mice exhibit striking defects in both spermatogenesis and oogenesis, which result in infertility. Keegan et al. (1996) reported that ATM is localized specifically to the synapsed chromosomes, a result that could not be duplicated by other laboratories (Barlow et al., 1998). Further experimentation is required to settle these contradictory results. A constant concern in immunocytological work is the reproducibility of staining patterns obtained with different antibodies and by different groups. Polyclonal antibodies tend to have individual specificities that sometimes are not conserved from one bleed to another. To minimize these effects, it is generally recommended that more than one antibody against the same antigen be used. Also, the antibody specificity should be determined in Western blots of cell extracts of the same type as the ones used in immunocytology, and in blocking experiments in which preincubation with the antigen should block the staining pattern generated by the antibody. The absence of ATM produces severe meiotic disruption in mice, with mislocalization of the RAD51/DMC1 and ATR complexes to the chromatin loops (Barlow et al., 1998), and leads to an arrest at the leptotene stage, which is partially reversed in Atm−/− p53−/− double mutant mice (Barlow et al., 1997b). Therefore, ATM may provide a checkpoint function necessary to monitor the events preceding RAD51 assembly on the chromosomes (e.g., the correct level of DSB formation and their resection) to ensure that they are correctly completed before RAD51 assembly is attempted (Barlow et al., 1998). Upon detection of DSBs, an ATM-dependent signal to prevent or delay meiotic progression is transmitted to downstream effector proteins such as p53 (Levine, 1997), probably by direct phosphorylation, and the meiotic progression is halted. In the absence of p53 protein (i.e., Atm−/− p53−/− double knockout), defective meiosis is probably allowed to proceed to a downstream checkpoint where an alternative, p53-independent surveillance pathway becomes functional. The spermatocytes of double knockout mice are thus arrested at pachytene. ATR is an essential component of surveillance mechanisms as indicated by its requirement for survival in human cells following DNA damage, and by the fact that Atr inactivation in mice results in early embryonic lethality (Dasika et al., 1999). The kinase activity of ATR is required during the S-phase of the cellcycle when replication is halted by hydroxyurea treatment, and at the G2/M transition to prevent mitosis in the presence of DNA damage (Cliby et al., 1998).

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Unlike ATM mutant cells, wild-type cells respond to ionizing radiation by inhibiting DNA synthesis. Overexpression of ATR can restore radiation-induced DNA synthesis in ATM−/− cells, arguing that ATR and AMT can substitute for each other in the DNA damage response (Cliby et al., 1998). It has been reported that ATR phosphorylates p53 in vivo and in vitro at a serine residue critical for the function of p53 as a tumor suppressor (Tibbetts et al., 1999). It is possible, therefore, that ATR signals the presence of DNA damage to the tumor suppressor p53, which then blocks cell cycle progression until DNA repair has been completed. During meiosis, ATR localizes to discrete foci along the chromosomal cores during early prophase I stages and it is still present on the SCs at pachytene (Moens et al., 1999). The majority of the ATR localization sites are distinct at the EM level from the RAD51 foci, implying that even if the two proteins may be involved in the same pathway of DNA repair, they do not interact directly. Interestingly, ATR foci are most abundant on the unpaired sections of the homologous chromosomes in the process of synapsis, and decrease in numbers on the synapsed cores where it persists until mid-pachytene. The unsynapsed regions presumably contain the unprocessed DSBs that did not yet interact with the homologous chromosome, and therefore may also identify chromosomal regions with delayed synapsis. This immunolocalization pattern suggests a role for ATR in monitoring not only the early steps of HR, but also the correct establishment of synapsis. Its function may be mediated by signals indirectly received from the RAD51/DMC1 complexes, given that these proteins identify the sites of yet unrepaired DSBs. ATR probably transduces these signals to effector proteins, such as p53, that have the ability to block meiotic progression if required. 2. RAD1 Human RAD1 (HRAD1) has been proposed to be a component of a DNA-damage checkpoint (Freire et al., 1998), based on its homology to the S. cerevisiae RAD17 and S. pombe Rad1+ genes (Lydall and Weinert, 1995; Al-Khodairy and Carr, 1992). Recent observations indicate that hRAD1 protein localizes in foci to both synapsed and unsynapsed chromosome cores, being most abundant during the early stages of prophase I, and that the number of foci decreases from leptotene to pachytene. The overall dynamics is similar to that of RAD51/DMC1, suggesting that the hRAD1-dependent checkpoint monitors the correct fulfillment of the early meiotic DSB processing, including the assembly of the strand exchange complexes. As in the case of ATR, at the EM level of resolution, the hRAD1 and RAD51/DMC1 proteins appear on distinct locations along the chromosome cores (Freire et al., 1998). This observation most likely implies that other proteins are required to bridge the gap between the site of a DSB (presumably identified by RAD51/DMC1 complexes) and the position of the sensor protein (hRAD1). Similarly to ATR, RAD1 may signal the status of DSB repair to effector molecules (Dasika et al., 1999) that delay or arrest meiotic progression.

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IV. Conclusions and Perspectives Repair and checkpoint proteins are not randomly dispersed in the chromatin loops, but form complexes specifically localized along the chromosome cores or SCs. This observation supports the hypothesis that SCs are required to stabilize the assembly of these proteins in close proximity to the DNA or even to facilitate their direct binding to the DNA. It is possible that the SCs are required to maintain the physical connections between the components of these complexes with a dynamic protein composition. As illustrated in Fig. 3, most of the proteins currently known to participate directly in meiotic HR or to monitor HR are abundant on the meiotic chromosomes during the early stages of prophase I (leptptene/zygotene), when DSB are created and the first steps in their repair are thought to occur (Padmore et al., 1991). The presence of checkpoint proteins (e.g., ATR and RAD1) in association with the meiotic chromosomes at these stages, as well as the early meiotic arrest in mouse spermatocytes lacking the surveillance protein ATM, suggests that the first steps in DSB processing are critical for their correct repair. The extensive molecular detail available for these early stages of meiotic HR is not found in the late steps of recombination, which include branch migration and resolution events. Many other genes are probably required for successful completion of meiotic recombination events in mammalian cells. Based on knowledge from the DNA repair field, it is expected that proteins found to be active in DNA damage repair in mammalian mitotic cells will also play some role in meiotic recombination. Meiosis-specific proteins acting directly in meiotic recombination or in its surveillance are also expected to be identified. Possible methods for their identification include complementation analyses with known meiotic yeast mutants, and twohybrid screens of testis or ovary cDNA expression libraries using proteins known to act in meiotic HR as baits. Characterization of the biochemical and cellular functions of these proteins, complemented with the definition of the complex functional interactions among them, represents the next step toward a full understanding of meiotic recombination mechanisms in mammalian cells.

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