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Michael Lichten, Ph.D.
Mechanism and Control of Meiotic Recombination
We study genetic recombination and chromosome structural changes that occur during meiosis and DNA damage repair, using budding yeast as a model system. Our goal is a description of the molecular steps of meiotic recombination, and how they are regulated in parallel with changes in chromosome structure and with cell cycle transitions that occur during meiosis. It is expected that this will also inform mitotic DNA damage repair and recombination.
Initiation of meiotic recombination
Meiotic recombination is initiated by double-strand DNA breaks (DSBs), formed by the type 2 topoisomerase paralog Spo11p in a mechanism that is universal among eucarotes. We used microarray-based analysis to map DSBs by an alternative method, which detects single-strand DNA associated with processed breaks in wild-type and in dmc1 mutants, which form and process DSBs but which do not repair them. We find that DSB sites are fairly evenly distributed across the genome, a finding consistent with Southern blot-based measures of DSB frequencies and with genetic measures of meiotic recombination (see picture 2 in Gallery).
As part of this project, we are developing general methods to isolate and analyze DSBs and other forms of DNA damage at the single nucleotide level, as well as analytical and computational tools to compare genome-wide distributions of events (such as DSBs) and to allow quantitative correlative comparison of distributions at different scales. These tools will be applied to genome-wide studies of chromosome composition and of differnt molecular events of chromosome metabolism, as well as to studies of the dynamics of these features over evolutionary time.
Partner choice in meiotic recombination
During meiosis, DSBs are frequently repaired by inter-homolog recombination, with the frequent production of Holliday junction-containing joint molecules (JMs) that are primarily precursors to crossovers (see below). In contrast, in mitosis the sister chromatid is the preferred partner, and JMs and crossovers are rare. Specialized chromosome structures called the synaptonemal complex are assembled during meiosis, including axial elements that coordinate sister chromatids. Axial element components and associated proteins are thought to control multiple aspects of meiotic recombination, most notably partner choice. In particular, in budding yeast, double-strand break-induced, axis-dependent signaling activates the Mek1 kinase, whose activity is required for the high level of interhomolog recombination seen in meiosis.
Using strains where a strong meiotic double-strand break forms in sequences present on only one of the two homologs, we showed that meiotic recombination between sister chromatids can occur at the same efficiency and with kinetics similar to that of recombination between homologs, consistent with the suggestion that a substantial fraction of meiotic DSBs are repaired by inter-sister recombination.
Inter-sister repair rates are increased about 3-fold in mek1 mutants, and interhomolog recombination is substantially reduced. Based on these findings, we suggest that a relatively modest reduction in the rate of inter-sister strand invasion, imposed at each step of the recombination process, helps to promote inter-homolog recombination during meiosis. We are testing this suggestion by characterizing early strand invasion intermediates, to see if partner choice biases are imposed at the initial step of meiotic recombination, at to identify the targets of Mek1 that participate in partner choice.
Regulation of meiotic recombination intermediate formation and resolution
The Holliday junction is thought to be a central intermediate in the recombination events that occur during DSB repair. We previously showed that the Holliday junction-containing joint molecules (JMs) that form during meiosis are predominantly precursors to crossover recombinants; most noncrossover recombinants arise by a different, earlier pathway. We are interested in identifying the activities resonsible for the formation and resolution of these intermediates, and the regulatory circuits that integrate these processes with the meiotic cell cycle.
We had previously shown that noncrossover and crossover recombination are regulated differently during meiosis. Mutants lacking NDT80 (a meiosis-specific transcription factor responsible for transcription of ~200 genes) or CDC5 (the yeast homolog of the mammalian polo-like kinase PLK1) arrest in meiosis with unresolved JMs and very few crossovers, while noncrossover heteroduplex products are formed at normal levels and with normal timing. Ndt80 is required for meiotic transcription of CDC5, and ectopic expression of CDC5 in ndt80 mutants restores JM resolution. Current efforts are aimed at identifying the targets of this kinases that are directly involved in recombination intermediate resolution as crossovers during meiosis.
Sgs1, the budding yeast homolog of the mammalian BLM helicase, has been suggested to have anti-recombination activity. Consistent with this, loss of the Sgs1 helicase restores crossover recombination and synapsis of homologous chromosomes to mutants lacking components of the synaptonemal complex, a meiosis-specific structure that tightly pairs homologous chromosomes and that is necessary for normal levels of crossover recombination (see picture 1 in Gallery). This identifies the Sgs1 helicase as a potent anti-recombinator whose action is blocked by meiosis-specific chromosome proteins and structures.
Mutants lacking both Sgs1 and the Mus81-Mms4 endonuclease, which has been suggested as a Holliday junction resolvase, accumulate unresolved recombination intermediates, many with structures that are infrequent in wild type cells. Using an inducible expression system, we restored Mus81 or Sgs1 to sgs1 mus81 cells at a time when JMs are forming. Mus81 expression did not prevent JM formation, but restored JM resolution, CO formation, and nuclear division. In contrast, Sgs1 expression reduced the extent of JM accumulation. These results indicate that Sgs1 and Mus81-Mms4 collaborate to direct meiotic recombination towards interhomolog interactions that promote proper chromosome segregation, and also indicate that Mus81-Mms4 promotes JM resolution in vivo.
Recombination intermediate resolution in the mitotic cell cycle
As a way to investigate recombination intermediate resolution during the mitotic cell cycle, we used a unique feature of S. cerevisiae, namely the ability exit meiosis and return to growth (RTG) upon nutrient restoration. Upon RTG, these cells abandon meiosis and switch to the mitotic cell cycle, and JMs that have accumulated are resolved in a mitotic environment. We find that, after RTG, intermediates are completely resolved, but most produce noncrossovers. In cells lacking the Sgs1 helicase, intermediate resolution is delayed until Cdc5 is expressed, and then resolution produces both crossovers and noncrossovers. Furthermore, this later resolution depends on Mus81-Mms4.
These findings indicate that, during the mitotic cell cycle, most JMs are resolved by Sgs1 helicase by mechanisms that do not produce crossovers. Cdc5 expression late in the cell cycle is associated with a switch in JM resolution, to Mus81-Mms4-dependent cleavage of JMs, producing both crossovers and noncrossovers. Current work is aimed at determining whether or not Cdc5 directly triggers this transition, and at identifying relevant Cdc5 targets.
This page was last updated on 12/2/2013.