The long-range objectives of our research are to understand the mechanism of meiotic recombination and to determine how this process is coordinated with other events of meiotic prophase. Several projects in the lab focus on Spo11 (the protein that makes the DNA double-strand breaks (DSBs) that initiate recombination), the proteins that interact with Spo11, the interactions of these proteins with meiotic chromosomes, and the mechanisms that regulate the timing, number, and location of DSB formation. We also study how DSBs are processed and repaired and we are seeking to expand knowledge of meiotic processes more generally through discovery of new genes important for germ cell function. Most of the meiosis research in the lab uses the yeast Saccharomyces cerevisiae and the mouse. Finally, our studies of Spo11 have also provided an entry point to research on the DSBs made by the enzyme Topoisomerase II when it is inhibited by chemotherapeutic agents such as etoposide or doxorubicin.
Meiosis is a specialized cell division that generates gametes in every sexually reproducing organism, from fungi to humans. During meiosis in most organisms, recombination helps homologous chromosomes pair and become physically connected, promoting accurate segregation. Recombination also alters genome structure by disrupting genetic linkage groups and, less often, by generating large-scale rearrangements between dispersed repetitive sequences. Meiotic recombination is thus a powerful determinant of germline genome stability, but also of genome diversity, evolution, and instability.
Meiotic recombination involves formation and repair of double-strand breaks generated by the Spo11 protein through a topoisomerase-like reaction in which a tyrosine severs the DNA backbone and attaches covalently to the 5’ end of the cleaved strand. Endonucleolytic cleavage liberates Spo11 bound to short oligonucleotides and resection yields 3’-single-stranded tails. These tails are substrates for strand-exchange proteins that catalyze invasion of homologous duplexes, ultimately giving rise to recombinant products. DSB repair can result in either reciprocal exchange of flanking chromosome arms (a crossover), or no exchange (a noncrossover). If there are sequence polymorphisms in the repair region, then formation of heteroduplex DNA and mismatch correction can yield nonreciprocal transfer of information from one chromosome to the other (gene conversion).
DSBs are hazardous genomic damage that most cells avoid, but that each meiotic cell introduces in large numbers (approximately 160 on average in yeast, approximately 200 to 300 in mice). Recombination usually occurs between allelic sequences on homologs or between sister chromatids, but can involve non-allelic DNA with high-sequence identity. Such non-allelic homologous recombination (NAHR) can cause chromosome rearrangements. Furthermore, outright failure of DSB repair can lead to meiotic arrest or lethal aneuploidy in gametes. To minimize the risk of deleterious effects, cells regulate Spo11 activity to occur only at the right time and right place, but mechanisms of this regulation remain poorly understood.
DSB formation by Spo11 is biochemically similar to action of type II topoisomerases, enzymes that normally work to create a transient gate in a DNA duplex by breaking both strands via a covalent protein-DNA complex that is then reversed to reseal the break. Topoisomerase II is a key player in both the etiology and treatment of many cancers, including both primary and metastatic diseases. The enzyme is a target of potent anti-cancer drugs such as etoposide, which trap the protein when it is bound to broken DNA and thus turn Topo II into a DNA-damaging toxin. Topo II can also promote tumor formation, as secondary leukemias occur in a fraction of patients treated with etoposide, and spontaneous Topo II errors are implicated in tumorigenic gross chromosomal rearrangements. However, despite widespread use of Topo II poisons in the clinic and the long-time appreciation that this enzyme has critical functions in genome integrity, remarkably little is known about how mammalian cells respond to and repair Topo II-generated DSBs. Our studies of Spo11 during meiosis have provided a foundation for studies of Topo II DSBs in mammalian cells.