Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation

Rad52 Associates with RPA and Functions with Rad55 and Rad57 to Assemble Meiotic Recombination Complexes

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes

Rad52 Associates with RPA and Functions with Rad55 and Rad57 to Assemble Meiotic Recombination Complexes

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes

Investigating the spatial regulation of meiotic recombination in S. cerevisiae

In order for a species to engage in and reap the evolutionary benefits of sexual reproduction, a subset of cells in each individual must undergo a complex ordeal known as meiosis—a specialised cell division. By halving the genome content and “shuffling the deck”, meiosis generates genetically diverse haploid gametes (eggs, sperm) or spores from diploid cells. Such a monumental task is by no means easy or risk free: during the meiotic programme, cells intentionally damage their own genomes through widespread induction of DNA double-strand breaks (DSBs) in order to initiate homologous recombination—a DNA-repair process—and subsequent crossover (CO) formation. The success of meiosis is, however, not left up to chance. Rather, a complicated web of regulation acts at multiple stages to ensure this dangerous tradeoff pays dividends. Notably, the spatial pattern of meiotic recombination across the genome is complex and non-random. Whilst ultimately stochastic in nature, recombination events within any given meiotic cell display relatively even distributions along each chromosome—a phenomenon mediate by processes of “interference” acting at two key stages in meiosis: DSB and CO formation. Despite wide ranging historical observation, relatively little is known about how either form of interference is accomplished. Genome-wide mapping of recombination within S. cerevisiae has, however, provided a unique opportunity to investigate the underlying mechanisms. By computationally and mathematically analysing genome-wide data, work presented throughout this thesis seeks to: (i) investigate CO distribution and CO interference within various DNA damage response and DNA repair mutants (Tel1ATM, Mec1ATR, Rad24, Msh2) (Chapter 2) (ii) develop novel approaches to DSB mapping (Chapter 3) (iii) characterise the hyperlocal regulation of DSB formation (Chapter 3) and (iv) examine the mechanics of DSB interference (Chapter 4). Moreover, widely applicable simulation platforms for investigating DSB and CO formation have been developed (Chapter 2, 4). Collectively, this thesis further elucidates the mechanisms that underpin the spatial regulation of meiotic recombination in S. cerevisiae

Protein Rates of Evolution Are Predicted by Double-Strand Break Events, Independent of Crossing-over Rates

Theory predicts that, owing to reduced Hill–Robertson interference, genomic regions with high crossing-over rates should experience more efficient selection. In Saccharomyces cerevisiae a negative correlation between the local recombination rate, assayed as meiotic double-strand breaks (DSBs), and the local rate of protein evolution has been considered consistent with such a model. Although DSBs are a prerequisite for crossing-over, they need not result in crossing-over. With recent high-resolution crossover data, we now return to this issue comparing two species of yeast. Strikingly, even allowing for crossover rates, both the rate of premeiotic DSBs and of noncrossover recombination events predict a gene's rate of evolution. This both questions the validity of prior analyses and strongly suggests that any correlation between crossover rates and rates of protein evolution could be owing to slow-evolving genes being prone to DSBs or a direct effect of DSBs on sequence evolution. To ask if classical theory of recombination has any relevance, we determine whether crossover rates predict rates of protein evolution, controlling for noncrossover DSB events, gene ontology (GO) class, gene expression, protein abundance, nucleotide content, and dispensability. We find that genes with high crossing-over rates have low rates of protein evolution after such control, although any correlation is weaker than that previously reported considering meiotic DSBs as a proxy. The data are consistent both with recombination enhancing the efficiency of purifying selection and, independently, with DSBs being associated with low rates of evolution

Interplay netween Dbf4-dependent Cdc7 kinase and polo-like kinase unshackles mitotic recombination mechanisms by promoting synaptonemal complex disassembly

Meiotic recombination is initiated by self-inflicted DNA breaks and primarily involves homologous chromosomes, whereas mitotic recombination involves sister chromatids. Whilst the mitotic recombinase Rad51 exists during meiosis, its activity is suppressed in favour of the meiosis-specific recombinase, Dmc1, thus establishing a meiosis-specific mode of homologous recombination (HR). A key contributor to the suppression of Rad51 activity is the synaptonemal complex (SC), a meiosis-specific chromosomal structure that adheres homologous chromosomes along their entire lengths. Here, in budding yeast, we show that two major cell cycle kinases, Dbf4-dependent Cdc7 kinase (DDK) and Polo-kinase (Cdc5), collaborate to link the mode change of HR to the meiotic cell cycle by. This regulation of HR is through the SC. During prophase I, DDK is shown to maintain SC integrity and thus inhibition of Rad51. Cdc5, which is produced during the prophase I/metaphase I transition, interacts with DDK to cooperatively destroy the SC and remove Rad51 inhibition. By enhancing the interaction between DDK and Cdc5 or depleting DDK at late prophase I, meiotic DNA breaks are repaired even in the absence of Dmc1 by utilising Rad51. We propose that the interplay between DDK and Polo-kinase reactivates mitotic HR mechanisms to ensure complete repair of DNA breaks before meiotic chromosomem segregation

Mutation Rates across Budding Yeast Chromosome VI Are Correlated with Replication Timing

Previous experimental studies suggest that the mutation rate is nonuniform across the yeast genome. To characterize this variation across the genome more precisely, we measured the mutation rate of the URA3 gene integrated at 43 different locations tiled across Chromosome VI. We show that mutation rate varies 6-fold across a single chromosome, that this variation is correlated with replication timing, and we propose a model to explain this variation that relies on the temporal separation of two processes for replicating past damaged DNA: error-free DNA damage tolerance and translesion synthesis. This model is supported by the observation that eliminating translesion synthesis decreases this variation

Contributions of the Human SSB Complex and MEI5-SWI5 Complex to Homologous Recombination

DNA double-strand breaks (DSBs) are the most threatening type of DNA damage in a cell. Homologous recombination (HR) is the most accurate repair mechanism for DSBs, and if HR fails, the integrity of the genome can be compromised. Two recombinases, RAD51 and DMC1, are vital for HR but require assistance for HR to proceed efficiently and accurately. Several proteins, including mediators, single-strand binding proteins, and accessory proteins, have been shown to function in the HR with the recombinases. Mediators are responsible for overcoming inhibition caused by the single-strand binding protein, Replication protein A (RPA). Accessory proteins assist the recombinases through DSB localization, ATP hydrolysis, filament stabilization and several other functions. In addition to RPA, higher eukaryotes possess two other SSBs, SSB1 and SSB2. Both hSSBs maintain genomic integrity through participation in the HR pathway. It was previously demonstrated that hSSB1 stimulates RAD51 during D-loop formation. Additionally, the hSSBs maintain genomic integrity through the repair of stalled replication forks. In this dissertation, we present in Chapter 2 surprising activities of the hSSBs that support the recent genetic data implicating hSSB1 and hSSB2 in the repair of stalled replication forks. We demonstrated a functional interaction with the human polymerase η in D-loop extension and second-end capture. This is the first report of the hSSBs interaction with a polymerase and identifies a new function of the hSSBs in DNA double-strand break repair. We also report that hSSB1 and hSSB2 can anneal single-strand DNA and melt double-strand DNA. In Chapter 3, we examined the effect of CaCl2 and MgCl2 on hSSB D-loop formation and demonstrate that hSSB1 and hSSB2 can in fact form D-loops in the absence of the recombinase, RAD51. Both hSSB1 and hSSB2 form a heterotrimeric complex with Integrator subunit 3 (INTS3) and the Single-strand interacting protein 1 (hSSBIP1). We have purified the components and confirmed complex formation. The effect of the complex proteins on D-loop extension by hPol η will be interesting to examine in the future. The hMEI5-SWI5 ortholog in Saccharomyces cerevisiae functions as a mediator to scDMC1. To date, there have been no reports regarding hMEI5-SWI5 functionality with hDMC1. In Chapter 3, we examined the DNA binding activity of hMEI5 and hSWI5 individually and as a complex (Mei5-Swi5), in addition to demonstrating physical interaction with both DMC1 and RPA. Importantly, we report that hMEI5 but not hSWI5 retains mediator activity to hDMC1 using an in vitro homologous DNA pairing assay. This is the first biochemical report on hMEI5-hSWI5

Genomic insights into fine-scale recombination variation in adaptively diverging threespine stickleback fish (Gasterosteus aculeatus)

Meiotic recombination is one of the major molecular mechanisms generating genetic diversity and influencing genome evolution. By shuffling allelic combinations, it can directly influence the patterns and efficacy of natural selection. Studies in various organisms have shown that the rate and placement of recombination varies substantially within the genome, among individuals, between sexes and among different species. It is hypothesized that this variation plays an important role in genome evolution. In this PhD thesis, I investigated the extent and molecular basis of recombination variation in adaptively diverging threespine stickleback fish (Gasterosteus aculeatus) to further understand its evolutionary implications. I used both ChIP-sequencing and whole genome sequencing of pedigrees to empirically identify and quantify double strand breaks (DSBs) and meiotic crossovers (COs). Whole genome sequencing of large nuclear families was performed to identify meiotic crossovers in 36 individuals of diverging marine and freshwater ecotypes and their hybrids. This produced the first genome-wide high-resolution sex-specific and ecotype-specific map of contemporary recombination events in sticklebacks. The results show striking differences in crossover number and placement between sexes. Females recombine nearly 1.76 times more than males and their COs are distributed all over the chromosome while male COs predominantly occur near the chromosomal periphery. When compared among ecotypes a significant reduction in overall recombination rate was observed in hybrid females compared to pure forms. Even though the known loci underlying marine-freshwater adaptive divergence tend to fall in regions of low recombination, considerable female recombination is observed in the regions between adaptive loci. This suggests that the sexual dimorphism in recombination phenotype may have important evolutionary implications. At the fine-scale, COs and male DSBs are nonrandomly distributed involving ‘semi-hot’ hotspots and coldspots of recombination. I report a significant association of male DSBs and COs with functionally active open chromatin regions like gene promoters, whereas female COs did not show an association more than expected by chance. However, a considerable number of COs and DSBs away from any of the tested open chromatin marks suggests possibility of additional novel mechanisms of recombination regulation in sticklebacks. In addition, we developed a novel method for constructing individualized recombination maps from pooled gamete DNA using linked read sequencing technology by 10X Genomics®. We tested the method by contrasting recombination profiles of gametic and somatic tissue from a hybrid mouse and stickleback fish. Our pipeline faithfully detects previously described recombination hotspots in mice at high resolution and identify many novel hotspots across the genome in both species and thereby demonstrate the efficiency of the novel method. This method could be employed for large scale QTL mapping studies to further understand the genetic basis of recombination variation reported in this thesis. By bridging the gap between natural populations and lab organisms with large clutch sizes and tractable genetic tools, this work shows the utility of the stickleback system and provides important groundwork for further studies of heterochiasmy and divergence in recombination during adaptation to differing environments

Meiotic spindle assembly checkpoint and aneuploidy in males versus females

The production of gametes (sperm and eggs in mammals) involves two sequential cell divisions, meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate to different daughter cells, and meiosis II resembles mitotic divisions in that sister chromatids separate. While in principle the process is identical in males and females, the time frame and susceptibility to chromosomal defects, including achiasmy and cohesion weakening, and the response to mis-segregating chromosomes are not. In this review, we compare and contrast meiotic spindle assembly checkpoint function and aneuploidy in the two sexes.Peer reviewe