MEIOSIS
Meiosis is essential for sexual reproduction. The formation of gametes requires reduction of the chromosome complement by half. A full chromosome complement is then restored by fertilization and creation of a zygote. While a number of useful comparisons can be made between the mechanics of mitotic and meiotic division, it is clear from the first meiotic division that the processes are not equivalent. In mitosis, homologous chromosomes (homologs) replicate to form connected sister chromotids, the chromosomes compact, and the sisters of the two homologs are drawn to opposite poles when the inter-sister connections lapse. However, in meiosis the homologs are drawn to opposite poles and the most obvious physical connection that lapses are the crossovers between the chromosomes; cytologically visualized as “Chiasmata”. This first meiotic division (meiosis I) reduces the homologous chromosome complement by half. The second meiotic division (meiosis II) occurs when the centromeric connections between sister chromosomes lapse and the individual chromotids segregate. With few exceptions, the genes and mechanism of meiosis appear identical in S.cerevisiae, S.pombe, C.elegans, Drosophila, Arabidopsis, Lilly, Pidgeon, mouse, rat, and human. Differences include the timing and structure of the synaptonemal complex relative to recombination between homologs as well as the genes that make up the synaptonemal complex. In the latter case, there is evidence that structure/function is conserved independent of amino acid sequence. We focus here on results from mouse and human studies.
The cytology of meiosis
Prior to meiosis chromosomes are duplicated to form two sister chromotids. After this premeiotic S phase, the chromosomes condense, and the centromeres and telomeres become attached to the nuclear envelope. The telomeres often cluster on the nuclear envelop and the chromosomes form a bouquet arrangement. It is at this time that a dense proteinaceous axial core or lateral element (LE) forms between chromotids. When the LE’s have been nearly completed, they are brought together in a “synapsis” process by the assembly of transverse filaments (TF) that ultimately result in the formation of an observable tripartite synaptonemal complex (two LE’s held by TF’s). The synaptonemal complex spans the entire length between the homologous chromosomes. While the complete complement of proteins associated with the synaptonemal complex is unknown, SCP2 and SCP3 (mouse/human) appear to be the major components of the LE’s and SCP1 (mouse/human; Zip1 in yeast) appears to be associated with the TF. During synapsis small dense proteinaceous nodules (early recombination nodule or ERN) appear between the homologous chromosomes. At pachytene many of these ERN’s resolve leaving one or two large late recombination nodules (LRN) per chromosome. The number and distribution of the LRN’s is consistent with the numbers and frequency of genetic recombination crossovers that are visualized in the microscope as Chiasmata. It is these crossovers that are the basis of genetic mapping discovered in the early 20th century.
Immunoflorescent protein localization and the formation of Chiasmata in meiosis I.
The physical alignment of homologous chromosomes that occurs in leptotene-zygotene and requires a double-strand break (DSB) repair event between homologs. The Spo11 gene product introduces a DSB on one homolog that is then resected by a 5’ to 3’ exonuclease (on both sides of the scission;. The 3’-end of one side is then used in a classic strand-invasion reaction onto the homologous chromosome. Strand invasion requires at least two eukaryotic proteins that are homologous to the bacterial recombination-initiation protein RecA: RAD51 and the meiosis-specific DMC1. Mutation of Spo11, RAD51, or DMC1 has been shown to result in a dramatic reduction of homologous chromosome pairing, a high frequency of meiosis I non-disjunction, and gamete inviability. Immunoflorescent analysis has been used to determine the timing and abundance of recombination protein association(s) with meiotic chromosomes (Figure 4). These results suggest that upward of 300 ERN’s are formed by RAD51/DMC1 beginning in leptotene. These ERN’s are used to align homologous chromosomes and form the synaptonemal complex. Once the synaptonemal complex is formed approximately 90% of these nascent recombination structures are resolved in a process that allows “gene conversion” but no “crossing over” between chromosome arms. The remaining 10% (25-30 in mouse) become LRN’s and chiasmata.
In S.cerevisiae, C.elegans, and mouse, mutation of either Msh4 or Msh5 results in meiotic defects and infertility. In a series of elegant experiments using a temperature sensitive C.elegans ceMSH4 mutant, Villeneuve and colleagues demonstrated that the meiotic defect was associated with the inability to form crossovers between homologs in pachytene of Meiosis I. This lack of crossovers resulted in a high frequency of meiotic nondisjunction (inappropriate chromosomal segregation). Genetic studies with similar yeast mutants have suggested that Msh4 and Msh5 are also intimately involved with both the formation and resolution of crossovers and can be visualized associated with "recombination nodules". Importantly, both the yeast and worm Msh4-Msh5 do not display a mutator phenotype, supporting the notion that they do not function in MMR.
Immunoflorescent localization of Msh4 suggests a timing and abundance similar to Rad51/DMC1 and ERN foci (Figure 5). Direct demonstration of colocalization has not been reported. Unlike Rad51/Dmc1, RPA, and BLM, the Msh4 foci persist into the LRN stage and appear to be fundamental components of the chiasmata. The LRN also contain MLH1 although colocalization of Msh4/Msh5 with Mlh1 has also not been demonstrated. Taken as a whole, the genetic and immunoflorescent data suggest a central role for MSH4-MSH5 in the formation and stabilization of chiasmata. Moreover, the absence of stable chiasmata results in chromosome non-disjunction and inviable gametes. Our biochemical studies exhibit remarkable integration with the available genetic and cellular data and suggest mechanism for hMSH4-hMSH5 in meiosis.
The role of hMSH4-hMSH5 in Meiosis I
We have developed a similar model for the role of hMSH4-hMSH5 in Meiosis I (Figure 6). In this model, Spo11 initiates meiotic recombination by inducing a double-stranded break (DSB) (Figure 6A). The ends of this DSB are resected in a RAD50-dependent manner leaving a 3’-ssDNA overhang (Figure 6B). This 3’-ssDNA may then be used as a substrate by hRAD51/hDMC1 in a classic homologous DNA strand-invasion reaction that forms a “D-loop” or “nacent Holliday Junction” (Pro-HoJo; Figure 6C). These nacent Holliday Junctions correspond to the approximately 300 foci observed on meiotic chromosomes. Multiple hMSH4-hMSH5 sliding clamps may form on the nacent Holliday Junction (Figure 6D). Such a stabilization event may be followed by a “primed DNA synthesis” that extends the D-loop until it may anneal to the distal end of the DSB (Figure 6E). It is at this point that a biological decision process occurs. In approximately 90% of the cases, these nacent Holliday Junctions dissociate and the overlapping ssDNA ends anneal in an apparent BLM1-mediated process (Figure 6F-G). This dissociation would physically correspond to the process of Cross-over Interference where DSBs retain the signature conversion events, associated with the primed DNA synthesis that used the homologous chromosome as a template, but do not display genetic crossing over. The remaining 10% of nacent Holliday Junctions are further stabilized by hMSH4-MSH5 and eventually become Chiasmata and genetic crossovers (Figure 6H-J). Our determination of hMSH4-hMSH5 function has solidified the Molecular Switch model for MMR while severely limiting the role of MMR proteins in homeologous recombination. It is also quite likely that we have one of the most complete collections of purified MMR and RR proteins.
Important Questions Of Meiosis I -- What triggers the release and segregation of homologous chromosomes at anaphase I? The above discussions clearly suggest that the complex processes of meiosis are being solved at the molecular level. However, several important questions remain. For example, is the function of hMSH4-hMSH5 merely to stabilize the chiasmata or is there some other fundamental role of hMSH4-hMSH5 in the chromosome segregation process? As suggested above some 300 homologous chromosome interactions induce homologous chromosome alignment and the formation of the synaptonemal complex. However, once formed the synaptonemal complex would in theory seem sufficient for the metaphase alignment and segregation of homologs in anaphase I. Why then retain one or two chiasmata per chromosome? One possibility suggests a more fundamental role for hMSH4-hMSH5 in the release of homologous chromosomes required for anaphase I. This additional function for hMSH4-hMSH5 would be comparable to cohesins in mitosis.
What is the molecular mechanism that leads to crossover interference? Crossover interference has been a major black box in genetics since it was discovered in the mid-twentieth century. The fundamental observation is that a single genetic crossover (exchange of genes across a chromosome length) interferes with the ability of a nearby crossover to occur. This interference can by transmitted for very long distances (10’s of megabases) along the length of a chromosome. Moreover, the signature of a “potential” crossover – gene conversion – can often be observed nearby a true crossover. Yet, these gene conversion events are always resolved to yield a non-crossover genetic condition. Mathematic analysis has suggested a “counting mechanism” in which a fixed number of “potential crossovers” are eliminated on either side of a true crossover. The molecular mechanism of crossover interference is unknown. Genetic studies have shown that MSH4-MSH5 strongly influences crossover interference. Yet, other than immunoflorescent localization of MSH4-MSH5 to chiasmata (observable crossovers), there is no obvious mechanism for these proteins in crossover interference.
PROJECTS
- Biophysical analysis of hMSH4-hMSH5; hMLH1-hMLH3
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