A fundamental question in biology is how the duplication, repair, and segregation of chromosomes are coordinated with each other and with progression through a cell division cycle. Homologous recombination during meiosis provides a fascinating case study for these processes. Meiosis is the specialized cell division that generates reproductive cells (sperm and eggs in mammals, spores in fungi). During meiosis, homologous maternal and paternal copies of each chromosome are separated from one another so that the reproductive cells end up with half of the starting genome complement.
In most sexual organisms, meiotic cells damage their own DNA and then repair the damage by exchanging genetic information between the homologous copies of each chromosome. This process, called homologous recombination, forms physical connections between homologs that allow them to segregate accurately. If recombination fails, chromosome segregation also frequently fails, with disastrous consequences. For example, recombination errors likely cause many instances of Down syndrome, in which a child inherits an extra copy of chromosome 21.
My laboratory studies how meiotic recombination works and how it is regulated so that it happens only at the right time and the right place. We focus principally on the yeast Saccharomyces cerevisiae and the mouse, with the latter largely in collaboration with Maria Jasin at Memorial Sloan-Kettering Cancer Center.
Controlling Meiotic Recombination Initiation
Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by the conserved Spo11 protein. This is a dangerous game: DSBs and the recombination they provoke are essential for meiosis, but DSBs are also potentially lethal genomic insults if repaired incorrectly or not at all. How do cells control DSB formation to foster its essential functions but minimize untoward effects?
One clue came from our discovery that cyclin-dependent kinase (CDK) promotes DSB formation in yeast. We showed that CDK phosphorylates Mer2 (one of nine yeast proteins required along with Spo11 for DSBs), thereby enabling interactions with other proteins needed for DSB formation. It is likely that CDK regulation of Mer2 coordinates recombination with meiotic progression. In ongoing work, we are testing our hypothesis that dual regulation of Mer2 by CDK and another kinase, Cdc7, also coordinates DSB formation with DNA replication.
Another clue came from our discovery that ATM, a kinase mutated in the cancer-prone disease ataxia telangiectasia, controls meiotic DSB numbers. Patients with this disease display gonadal dysgenesis and Atm-deficient mice are sterile because ATM is essential during meiosis, but it was unclear what specific roles it plays. We found that mice lacking ATM make many more Spo11-generated DSBs than normal and that this increase in DSBs is responsible for many (but not all) of the meiotic defects in the absence of this kinase. We inferred that an essential function of ATM is to govern a negative feedback loop that restrains Spo11 to limit DSB numbers.
This finding was intriguing. While it has long been known that different organisms experience widely different DSB numbers (ranging from dozens per cell in nematodes, to hundreds in yeast and mammals, to thousands in lilies), little was known about the regulatory pathways controlling these species-specific DSB setpoints. ATM is clearly part of this regulation. More recently, we uncovered evidence of other pathways in yeast and mice that act as feedback mechanisms linking DSB number with successful interactions between homologous chromosomes. This linkage explains how self-organizing meiotic chromosomes can continue making DSBs where they are needed (i.e., where interhomolog interactions have not yet come about) and shut down Spo11 where additional DSBs are no longer necessary.
The Landscape of Meiotic Recombination
Recombination alters genome structure by disrupting genetic linkage groups, so it is a powerful determinant of diversity and evolution. Recombination occurs more often in some places than others, largely because of nonrandom DSB distributions that display many levels of spatial organization. There are large DSB hot and cold domains, within which are short regions, called hotspots, where DSBs form preferentially. Important determinants of this organization include open chromatin structure and the presence of certain histone modifications. However, detailed understanding of how these and other factors influence DSB locations is lacking.
We developed a novel method to map DSBs, taking advantage of a quirk in the recombination mechanism. Spo11 breaks DNA via a topoisomerase-like reaction, generating a covalent protein-DNA intermediate. We proposed and then proved that Spo11 is removed from DSB ends by endonucleolytic cleavage, releasing Spo11 covalently attached to a short oligonucleotide. These oligonucleotides are sequence tags that mark where Spo11 made a DSB. We deep-sequenced Spo11 oligonucleotides from yeast, and the resulting map improved the spatial resolution of existing maps by some two to three orders of magnitude.
Our analysis of this map supports the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. This map also illuminated the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We are now exploiting Spo11 oligonucleotide mapping to study changes in DSB patterns in mutants with defects in chromosome structure and are extending this methodology to mice, fission yeast, and other organisms.
Controlling Recombination Outcome
Each chromosome pair forms at least one crossover, despite a low average number of crossovers per chromosome, and multiple crossovers tend to be evenly and widely spaced. Aspects of this "crossover control" have been recognized for nearly a century, since pioneering studies in Drosophila by Hermann Muller and colleagues. The mechanisms involved are not well understood. More DSBs are formed than crossovers, so crossover control involves a decision by which a subset of DSBs becomes crossovers, while all other DSBs follow a pathway(s) that generates primarily noncrossover products. To understand the logic of this decision, we examined recombination in yeast when breaks are reduced by hypomorphic spo11 mutations. We found that crossovers tend to be maintained at the expense of noncrossovers, a previously unsuspected manifestation of crossover control that we call crossover homeostasis. We have extended these studies to demonstrate homeostatic crossover behavior in mice as well, and we are using crossover homeostasis and genetic and physical assays that measure this phenomenon to explore the mechanism of crossover control.
These studies are funded in part by grants from the National Institute of General Medical Sciences and the National Institute of Child Health and Human Development.
As of April 08, 2013