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Regulating Joint Molecule Intermediates During Homologous Recombination
Summary: Neil Hunter uses yeast and mice to study homologous recombination, an essential chromosome repair process.
Faithful propagation and preservation of the genome depends on the process of homologous recombination, in which a broken or damaged chromosome utilizes a second chromosome as a repair template. Homologous recombination is an intrinsic part of the meiotic program, where it facilitates the pairing and segregation of homologous parental chromosomes. Moreover, by creating new allele combinations, meiotic recombination plays a fundamental role in genome evolution. The essence of recombination is the pairing and strand-exchange reaction that occurs between a free DNA end and a homologous template chromosome. The resulting joint molecule intermediate creates a primer for DNA synthesis in order to copy lost or damaged sequences. Finally, the involved chromosomes are dissociated by resolving the joint molecule. Resolution can occur with one of two outcomes: a crossover, in which chromosome arms are exchanged, or a noncrossover, involving only a local alteration of DNA.
In somatic cells, aberrant recombination causes chromosomal alterations that may activate oncogenes, cause loss of heterozygosity for tumor-suppressor genes, and ultimately lead to transformation and tumorigenesis. In meiotic cells, defective recombination is linked to infertility, miscarriage, and genetic diseases such as Down syndrome. It follows that recombination is tightly regulated at multiple levels. In this respect, somatic and meiotic cells are strikingly different, despite the fact that they utilize the same core recombination machinery. In somatic cells, for example, recombination is employed sporadically to repair chromosome damage and to restart stalled and broken replication forks. In contrast, meiotic cells induce recombination as a programmed event by inflicting hundreds of DNA double-strand breaks (DSBs) throughout the genome. To maintain recombination fidelity, somatic cells preferentially utilize the sister-chromatid template and actively suppress the crossover outcome. In meiotic cells, however, a homolog template must be engaged and at least one crossover forms between each pair of chromosomes to ensure accurate segregation at the first meiotic division. In all cell types, recombination must be coordinated and integrated with the other events of the cellular program.
My long-term goal is to ascertain how recombination is regulated to produce the outcome most appropriate to the cellular context, i.e., a regulated distribution of crossovers during meiosis and accurate repair in mitotically cycling cells. We are utilizing two model organisms: budding yeast and mouse. In budding yeast, my lab uses specialized molecular techniques to detect and monitor joint molecules formed in vivo. These powerful approaches provide mechanistic insights that are not revealed by more conventional methods. Studies in mouse take advantage of the superb meiotic cytology developed in this organism and are directly relevant for understanding recombination in humans.
Joint Molecule Metabolism During Meiotic Recombination
Meiotic cells form hundreds of DNA DSBs, but only a fraction (10–30 percent) result in crossover recombination. Moreover, the crossovers that do form show a highly regulated distribution: each pair of chromosomes almost always obtains at least one crossover, as required for accurate segregation, even though the total number of crossovers is typically very low (one to a few per chromosome). At the molecular level, we have provided evidence that the crossover or noncrossover fate of a recombination event is assigned and implemented via the differential stabilization and resolution of joint molecule intermediates. In budding yeast, two prominent types of joint molecule have been identified in vivo. Strand invasion by one end of a DSB gives rise to a single-end invasion. Subsequent interaction with the second DSB end, together with recombination-associated DNA synthesis, leads to formation of a double Holliday junction.
We are investigating three aspects of meiotic joint molecule regulation: (1) template choice: joint molecules form preferentially between homologs rather than sister-chromatids; (2) crossover/noncrossover differentiation: only crossover-designated recombination events form stable joint molecule intermediates; (3) joint molecule resolution: double Holliday junction resolution must be biased to produce a crossover outcome, and all joint molecules must be completely dissociated to restore duplex continuity and permit chromosome segregation.
The Roles of Post-Translational Protein Modification in Meiotic Recombination
Several types of covalent modification, such as phosphorylation and ubiquitylation, are employed to regulate protein activities. We know little about the roles of these post-translational protein modifications during meiosis, but recent studies in both yeast and mouse point to a critical role. We are analyzing known recombination proteins for post-translational modification during meiosis. This project is currently focused on parallel investigations of the putative SUMO (small ubiquitin-like modifier) E3 ligases, yeast Zip3 and mammalian Rnf212. Understanding the role of the mammalian Zip3 homolog, Rnf212, is of special interest because allelic variants in the RNF212 gene were recently associated with changes in genome-wide meiotic recombination rates in the human population. Moreover, higher recombination rates positively correlate with higher fecundity in humans. Thus, understanding the role of Rnf212 in mammalian meiosis will inform our understanding of human fertility.
Joint Molecule Metabolism During Mitotic DSB Repair
The discoveries we have made in meiotic cells have implications for homologous recombination in mitotically cycling cells. A major impediment to studying the molecular events of mitotic recombination in vivo has been the inability to detect and monitor joint molecules. Using a specially constructed molecular assay, similar to that used in our meiotic studies, my lab has now identified joint molecules formed during the repair of DSBs in mitotically cycling cells.
Our assay system has two important features: (1) it utilizes diploid cells in which both homolog and sister-chromatid templates are available, thereby making it a better model for recombination in (diploid) human cells; (2) the frequency of DSB formation is sufficiently low that both sister chromatids are almost never broken at the same time. This leaves the preferred sister-chromatid template intact and available for repair and thus reflects the normal physiological mode of DSB repair. The ability to monitor joint molecules formed in vivo during mitotic DSB repair will allow us to address gaps in our understanding of the mechanism and regulation of this process.
These studies are funded in part by grants from the National Institute of General Medical Sciences.
Last updated September 01, 2009
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