Preservation and propagation of the genome depends on homologous recombination, a template-dependent chromosome repair process. Recombination is also essential for the precise halving of the chromosome complement that occurs during meiosis to produce gametes (sperm and eggs). By creating new combinations of parental gene alleles, meiotic recombination also fuels evolution. Defective recombination during meiosis is linked to infertility and pregnancy miscarriage, and is a leading cause of congenital disease.
At the heart of recombination is the DNA pairing and strand-exchange reaction that occurs between the ends of a chromosome break and a homologous template chromosome (Figure 1). The resulting joint-molecule intermediate creates a primer-template substrate for the new DNA synthesis that is required to rejoin the broken chromosome. Ultimately, engaged chromosomes must be dissociated by resolving the joint molecule. Resolution can be associated with a crossover, in which chromosome arms are exchanged, or a noncrossover without exchange. During meiosis, crossovers combine with connections (cohesion) between sister-chromatids to tether homologous chromosome pairs allowing their stable biorientation on the spindle, which guides faithful segregation at the first meiotic division (Figure 2).
Meiotic cells initiate hundreds of recombination events via programmed DNA double-strand breaks, but only a fraction result in crossovers. Importantly, the crossovers that do form show a highly regulated distribution: each pair of chromosomes always obtains at least one crossover, as required for accurate segregation, even though the total number of crossovers is very low (one or two per chromosome). Notably, crossovers are not confined to specific sites, but can occur more or less anywhere along the chromosomes. These features make this "crossover assurance" a unique regulatory process in chromosome biology.
The fundamental research performed in the Hunter lab aims to delineate the molecular mechanisms of crossing over and how this process is regulated to ensure faithful chromosome segregation during meiosis. Our research utilizes two model organisms: budding yeast and mouse. In budding yeast, we exploit specialized techniques to monitor joint molecules formed in vivo (Figure 3) and apply “real-time genetics” to dissect the molecular machinery of recombination (Figure 4). These powerful assays provide mechanistic insights that are not revealed by more conventional approaches. Moreover, the highly conserved nature of meiotic recombination makes our discoveries relevant for understanding meiosis in humans. Our mouse studies take advantage of the superb meiotic cytology developed for this organism (Figure 5), the ability to study sexually dimorphic aspects of meiosis and gametogenesis, and direct relevance for understanding common pathologies of human meiosis
Formation and Resolution of Joint Molecules
How crossover sites are selected and how the molecular events of crossing-over are subsequently implemented at these sites remain outstanding questions. Molecular analysis in budding yeast indicates that crossovers and noncrossovers form via distinct pathways: crossing over involves joint molecules called double Holliday junctions (Figure 6), while noncrossovers are inferred to arise via transient D-loops (Figure 1). Thus, the fate of a recombination event is assigned and implemented by the differential stabilization and resolution of joint molecule intermediates. We are investigating several aspects of meiotic joint molecule metabolism including:
(1) Differentiation of crossover and noncrossover pathways. Current studies are focused on meiosis-specific factors that regulate the stability of joint molecules in particular the MutSγ complex comprising Msh4 and Msh5 – two meiosis-specific homologs of the DNA mismatch recognition factor, MutS. MutSγ is selectively stabilized at designated crossover sites and is subject to multiple levels of regulation by post-translational protein modification.
(2) The nature and regulation of recombination-associated DNA synthesis. Local DNA synthesis must accompany all recombination events. The nature and regulation of this special DNA synthesis during meiosis remains vague. For example, does only uncoupled leading-strand synthesis occur; which replication factors are required; how are replication factors recruited to nascent joint molecules; and how is recombination associated DNA synthesis regulated?
(3) Resolution of joint molecules. Joint molecule resolution must achieve two biological imperatives – biased resolution of double Holliday junctions to produce at least one crossover between every pair of chromosomes; and efficient and timely resolution of all joint molecules to allow chromosomes to separate. We have identified five nuclease activities that account for essentially all joint molecule resolution during meiosis, including a crossover-specific factor comprising the DNA mismatch-repair factors, Exo1 and MutLγ. Our recent studies also reveal that topological organization and entanglement of DNA are critical aspects of joint molecule processing, as revealed by the essential roles of SMC proteins and topoisomerases. Current analysis is extending these studies to determine the relationships between chromatin organization, topoisomerases and endonucleases during meiotic joint-molecule resolution.
Crossover Control by Regulated Protein Destruction
Covalent modifications, such as phosphorylation and ubiquitylation, are employed to regulate protein activity and turnover via proteasomal destruction. We know little about the roles of such post-translational protein modifications in regulating meiotic recombination, but studies in a number of model systems point to a critical role. Our recent studies have revealed central roles for the ubiquitin-family protein, SUMO, in regulating meiotic recombination. In mouse, designation of crossovers involves selective localization of the SUMO ligase RNF212 to a minority of recombination sites (Figure 7), where it stabilizes pertinent recombination factors such as MutSγ. This crossover/non-crossover differentiation process requires HEI10, an ubiquitin E3-ligase that antagonizes RNF212, promoting its removal from synapsed chromosomes and allowing recombination to progress. Together, these studies suggest a model in which the crossover or noncrossover fate of a recombination event is dictated by differential stabilization of recombination factors; with SUMO and ubiquitin playing antagonistic roles that are balanced to regulate the number and distribution of crossovers.
Ongoing investigations aim to identify pertinent targets of SUMOylation and ubiquitylation in both mouse and yeast, and to better understand the relationship between RNF212 and HEI10. These studies include genetic and biochemical analysis of SUMO and ubiquitin conjugation, and development of a pipeline to monitor protein SUMOylation during meiosis and map modified sites with high efficiency.
Crossing Over and Human Genetics
Understanding the functions of RNF212 and HEI10 are of special interest because human population studies have linked variants in the Rnf212 and Hei10 genes to changes in genome-wide crossover rates. Moreover, higher recombination rates positively correlate with higher fecundity in human females and advancing maternal age appears to select for oocytes with elevated crossover numbers. We have initiated studies of other genes linked to crossover rate by human population studies to determine whether they play direct roles in meiosis and understand their potential impact on human reproduction.
These studies are funded in part by grants from the National Institute of General Medical Sciences.
As of May 3, 2016