MEIOSIS

 


Figure 1. Illustration of Meiosis. Adapted from Marston and Amon, (2004)

 


Figure 2.  Immunofluorescence Protein Localization (IPL) of Meiosis I DSB Repair Proteins. IPL from mouse testis samples.  (A) Rad51, (B) RPA, (C) hMSH4, (D) hMSH5, (E) hMLH1, (F) hMLH3.  Stages include Leptotene (Lept), early Zygotene (EZ), late Zygotene (LZ), early Prophase (EP), middle Prophase (MP), late Prophase (LP).  Bottom panels are representative IPL for hMSH4 (upper) and hMSH5 (lower) in early and late prophase (LàR).  Synaptonemal complex protein SYCP3 (green), hMSH4 or hMSH5 (red) and centromeric protein CREST (blue)

All sexually reproducing organisms undergo meiosis: a process that reduces the cellular diploid content to produce haploid gametes. Meiosis begins with replication that forms sister chromosomes (chromatids) and is followed by a pairing process that spatially associates chromosome homologs (Fig. 1).  The segregation of chromosome homologs is completed in the first meiotic division (meiosis I) and the segregation of chromatids is completed in the second meiotic division (meiosis II); ultimately producing haploid gametes.  The mechanism, regulation, and checkpoint functions of meiosis II appear similar to the well-defined processes associated with mitosis.  In contrast, meiosis I requires the suppression of the tendency to segregate sister chromatids and instead the homologous chromosomes are separated.  More than 50% of all spontaneous miscarriages are due to errors in chromosome segregation (non-disjunction) at the first meiotic division.  Moreover, 90% of Down syndrome cases can be attributed to errors in maternal meiosis. With few exceptions, the critical meiosis genes appear identical in all eukaryotes.

 

Protein localization in meiosis I – The pairing of homologous chromosomes is complex process fraught with many pitfalls.  Chromosome pairing is initiated in Prophase I (Fig. 1) by the Spo11 gene productwhich actively introduces DNA double stranded breaks (DSBs) into the sister chromatids.  The landscape and number of meiotic DSBs appears dependent on localized chromatin structure and the ATM damage checkpoint process.   The repair of these DSBs by the nearest sister is suppressed by the formation of meiosis-specific lateral elements between the chromatids.  This leaves the homologous chromosome as the usual sequences exploited for DSB repair to restore the integrity of the genome.  The DSBs are ressected by a 5’à3’ exonuclease.  The resulting 3’ single-stranded DNA (ssDNA) end is then used in a search, pairing and strand-invasion reaction with the homologous chromosome that requires RAD51:  a homolog to the prototypical bacterial recombination-initiation protein RecA.  The ssDNA binding protein RPA is an essential RAD51 cofactor.  Mutation of Spo11 or RAD51 result in a dramatic reduction of homologous chromosome pairing, a high frequency of meiosis I non-disjunction, and gamete inviability. Immunofluorescent protein localization (IPL) has been used to determine the timing and abundance of recombination protein(s) on meiotic chromosomes (Fig. 2).  These results suggest that upward of 400 DSB sites are formed that contain RAD51 and RPA beginning in leptotene (Fig. 2A and 2B).  

 

Approximately 90% of the DSB repair events are resolved to form “gene conversion” signatures.  The remaining 10% (40-50 in human) become visible chromosomal crossovers known as chiasmata.  Ultimately, there are two significant events associated with meiotic DSB repair:  1.) genetic information is exchanged between chromosomes that is the basis of modern genetics, and 2.) homologous chromosomes become linked via chiasmata (Fig. 1).  

 

Mutation of the MutS homologs MSH4 or MSH5 results in defective meiosis and infertility that includes the inability to form chiasmata, which results in a high frequency of meiotic nondisjunction.  Importantly, yeast, worm and mouse MSH4-MSH5 do not display a mutator phenotype, supporting the notion that they do not function in mismatch repair (MMR) where most MutS and MutL homologs operate.  IPL studies indicate that MSH4 and MSH5 arrive later than Rad51/RPA; but at an equivalent abundance (Fig 2C and 2D).  However, unlike Rad51/RPA, some MSH4 and MSH5 foci persist and appear to be fundamental components of the chiasmata.  These chiasmata eventually contain the MutL homologs MLH1 and MLH3 (Fig 2E and 2F).  The genetic and IPF data suggests a central role for MSH4-MSH5 in the progression to the formation of a chiasmata. 

 


Figure 3. Model for the Deposition of Core Meiosis I Recombination Proteins.

Data from a number of organisms have demonstrated that the chiasmata begins as a D-loop DSB repair intermediate that then progress into linked double Holliday Junctions (HJ).  HJs contain crossovers of individual DNA strands that may be exchanged for long distances by the process of branch migration.  However, persistent HJs would evoke enormous topological constraints during chromosome segregation.  We proposed a model that would eliminate these topological issues, where MSH4-MSH5 formed sliding clamps that link homologous chromosomes (Fig. 3).  Such a linkage would preserve the chiasmata while allowing the resolution of the crossover DNA strands of the HJ (Fig. 3G).  This model is also consistent with IPL studies (Fig. 2) and has suggested a functional interaction between RAD51, MSH4-MSH5, MLH1-MLH3 and HJs that ultimately form the chiasmata.  

 

Questions:

 

1. How is RAD51 removed from D-loop intermediates?   RAD51 forms a nucleoprotein filament (NPF) that is required for homologous pairing and strand exchange (recombinase) functions.  While there appear to be several proteins including BRCA2 that help to load a RAD51 onto nascent recombination structures, no cofactors have been identified that fully disassemble the RAD51 NPF following recombination. 

 

2. Does MSH4-MSH5 recognize D-loop progenitor Holliday Junction intermediates formed by RAD51? Because RAD51 remains tightly bound to the D-loop recombinase product and disassembly of RAD51 results in dissociation of the D-loop, there appear to be only two possibilities for subsequent MSH4-MSH5 interactions:  i.) some other protein stabilizes RAD51 D-loops prior to MSH4-MSH5 recognition and sliding clamp formation, or ii.) MSH4-hMSH5 recognizes D-loops bound by RAD51.

 

3. Does a branch migrating Holliday Junction influence MSH4-hMSH5 recognition?   And a related question: Does MSH4-hMSH5 binding alter Holliday Junction branch migration?  It is not presently clear whether conversion tracts and HJ resolution that result in the signatures of genetic exchange are merely a consequence of, or are actively regulated by, the meiosis chromosome pairing mechanism.  Conversion tract length depends on the branch migration distance from the D-loop initiation site.

 

4. Does the interaction of MLH1-hMLH3 with MSH4-MSH5 alter the Holliday Junction or its physical properties?   Chiasmata uniquely contain MSH4-MSH5 and MLH1-MLH3, most likely in a complex.  The effect of this complex on HJ mechanics is unknown.  Moreover, the MLH3 subunit contains a cryptic endonuclease domain that in other MLH heterodimers has been shown to display endonuclease activity.

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