Molecular Genetics of Meiotic Recombination

 

Meiotic recombination serves several important functions. For example, recombination events mature into chiasmata, ensuring the proper segregation of homologous chromosomes during the first, or reductional, meiotic division. In the absence of recombination, homologous chromosomes do not assort properly, resulting in aneuploidy and usually death of the embryo. In certain cases, these aneuploids survive; in humans, this results in syndromes such as Down’s, Turner’s and Klinefelter’s. Our long-term goals are to understand the mechanism, regulation, and genetic controls of meiotic recombination and chiasmata in D. melanogaster. In order to study meiotic recombination in a genetic system, we are analyzing recombination defective mutants in Drosophila melanogaster.

Based on the analysis of several meiotic mutants, our current understanding of the meiotic recombination pathway in Drosophila can be briefly summarized. First, pairing of homolgous chromosomes culminates in synapsis, where they are tightly held together by a meiosis-specific structure, the synaptonemal complex (SC). Synapsis of homologs requires the c(3)G gene product. Next, the initiation of recombination requires both of the mei-W68 and mei-P22 gene products to generate DSBs. Repair of the DSBs creates recombination intermediates like the Holliday junction. General and meiosis-specific DSB repair genes such as mei-41 and spnB are required for this process. Finally, these intermediates are resolved into crossovers, a process that requires the mei-9 and mei-218 genes.

 

Direct Observation of Double Strand Breaks Sites During Meiosis

Meiotic recombination is initiated with a double-strand break (DSB). mei-W68 encodes a protein which most likely is the enzymatic factor which makes the double strand break. Yet it is clear that this enzyme is not sufficient; several proteins are required to make a meiotic double strand break. For example, the synaptonemal complex is required for DSB formation. In addition, studies in yeast and our lab have shown that additional proteins are required to enable MEI-W68 to make a break. One of these genes in Drosophila is mei-P22. We have cloned mei-P22 and found that it is associated with chromosomes early in meiotic prophase. This for the first time (in any organism) enables us to directly observe DSB sites in the cell. Used in conjunction with an antibody to the SC we have shown that DSBs occur after SC formation. This confirms previous genetic studies and shows there is a fundamental difference between budding yeast and some metazoans in how meiotic recombination is regulated.

 

DSB Repair and Resolution into Crossover Products

Once the double strand break forms, several events follow which result in the production of crossovers. The conventional view is that DNA repair factors bind to the DSB site and stimulate repair by a recombination repair mechanism using the homolog as a template. But there are also chromatin changes. In response to a double strand break a variant Histone H2A, H2AvD, is phosphorylated. Using an antibody first used in mammalian cells, we have detected this modification in Drosophila meiotic cells. Our studies show that at the sites of DSBs phophorylated H2AvD accumulates and then rapidly disappears. Its disappearance correlates with DNA repair. In mutants that are defective in DNA repair, such as RAD51 and RAD54 homologs, H2AvD phophorylation persists longer in meiosis.

The most complex and poorly understood process is how the repair event results in a crossover. In the standard view repair of the DSB during meiosis results in the formation of a double Holliday junction, in which the homologs are engaged in reciprocal strand exchange reactions, and this is required for crossing over to occur. The behavior of mutants defective in DSB repair is consistent with this model. We have shown that double strand break repair mutants are able to repair the DSBs, although at a slower rate than in wild-type. A more pronounced effect of these mutants, however, is that crossing over is drastically reduced. Therefore, the formation of crossovers requires a specific type of DSB repair.

A different type of crossover defective mutant is the mei-218 class, in which DSB formation and repair proceeds normally, but crossing over is drastically reduced. Thus, these genes are required specifically for the resolution of the recombination intermediate (i.e. Holliday junction) into crossovers. We have found that the MEI-218 protein is in the cytoplasm, which suggests it has a regulatory rather than a direct role in meiotic recombination. Genetic studies of this mutant have challenged the original DSB repair model that gene conversion and crossing over were alternative resolutions of the same intermediate. In fact, a different intermediate may exist for each outcome. These results suggest that the alternative outcomes, gene conversion or crossover, result from different repair and resolution reactions. Thus, crossing over may require a radically different set of proteins and specific events to occur. The idea that crossovers arise by a different mechanism than gene conversions explains the phenotype of our strong crossover defective mutants like mei-218.

 

A Kinesin Motor Protein Required for Homolog Segregation at Meiosis I 

Meiotic spindles in the oocytes of many species form without centrioles. In these systems the mechanism by which spindle poles form is not known (see Figure). We have recently identified a gene (Dub) that is required for the final aspect of meiotic crossover function, the segregation of homologs. In this mutant, chromosomes that were engaged in a crossover and would normally segregate at meiosis I, fail to do so. We have cloned this gene and found it encodes a kinesin-like protein. These proteins are microtubule dependent motor proteins. Our cytological analysis of this mutant has shown that this gene is required for spindle assembly and function. In particular, in the mutant there is a defect in spindle pole formation. In the absence of two well define spindle poles, chromosomes are not segregated correctly into two daughter nuclei. We are continuing the analysis of this gene, which will provide new insights into the function of the meiotic spindle, the mechanism of spindle pole formation, and how it interacts with the chromosomes to ensure segregation of the homologs.

 

 

Figure legend: A model for spindle pole formation in oocytes that lack centrosomes. The chromosomes substitute for the centrosomes by capturing microtubules. These initially do not form into a bipolar array. Shaping two poles is accomplished by the action of at least two motor proteins, which link two microtubules and move towards their minus ends. This “bundling” activity will bring together the array of microtubules into a defined point.

 

Lab Members

Dr. Kim S. McKim

Assistant Professor

 

Janet Jang

Research Associate

 

Hao Liu

Graduate Fellow

 

Elizabeth Manheim

Graduate Fellow

 

Rajal Patel

Laboratory Technician

 

Dalia Perelmuter

Laboratory Technician

 

Kelley Giunta

Undergraduate Student

 

Andrew Yanofsky

Undergraduate Student