Meiotic recombination involves the formation and repair of programmed DNA double-strand breaks (DSBs) catalyzed by the conserved Spo11 protein. This review summarizes recent studies pertaining to the ...formation of meiotic DSBs, including the mechanism of DNA cleavage by Spo11, proteins required for break formation, and mechanisms that control the location, timing, and number of DSBs. Where appropriate, findings in different organisms are discussed to highlight evolutionary conservation or divergence.
It has been long appreciated that, during meiosis, DNA replication is coordinated with the subsequent formation of the double-strand breaks (DSBs) that initiate recombination, but a mechanistic ...understanding of this process was elusive. We now show that, in yeast, the replisome-associated components Tof1 and Csm3 physically associate with the Dbf4-dependent Cdc7 kinase (DDK) and recruit it to the replisome, where it phosphorylates the DSB-promoting factor Mer2 in the wake of the replication fork, synchronizing replication with an early prerequisite for DSB formation. Recruiting regulatory kinases to replisomes may be a general mechanism to ensure spatial and temporal coordination of replication with other chromosomal processes.
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•Local replication timing dictates meiotic DSB timing (replication-DSB coordination)•Tof1/Csm3 recruits DDK to moving replication forks to promote Mer2 phosphorylation•Mer2 phosphorylation in the fork’s wake triggers recruitment of other DSB proteins•Replication-associated phosphorylation establishes replication-DSB coordination
To ensure that DNA replication during meiosis is followed by recombination events, a kinase that can promote the formation of double-strand breaks is recruited to the replisome. Consequently, the DNA breaks that initiate recombination are primed to form in the wake of the replication fork.
The nonrandom distribution of meiotic recombination shapes heredity and genetic diversification. Theoretically, hotspots—favored sites of recombination initiation—either evolve rapidly toward ...extinction or are conserved, especially if they are chromosomal features under selective constraint, such as promoters. We tested these theories by comparing genome-wide recombination initiation maps from widely divergent Saccharomyces species. We find that hotspots frequently overlap with promoters in the species tested, and consequently, hotspot positions are well conserved. Remarkably, the relative strength of individual hotspots is also highly conserved, as are larger-scale features of the distribution of recombination initiation. This stability, not predicted by prior models, suggests that the particular shape of the yeast recombination landscape is adaptive and helps in understanding evolutionary dynamics of recombination in other species.
Meiotic recombination is carried out through a specialized pathway for the formation and repair
of DNA double-strand breaks made by the Spo11 protein, a relative of archaeal topoisomerase VI. This
...review summarizes recent studies that provide insight to the mechanism of DNA cleavage by Spo11, functional
interactions of Spo11 with other proteins required for break formation, mechanisms that control the timing
of recombination initiation, and evolutionary conservation and divergence of these processes.
The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and ...distribution. Mechanisms of this regulation remain poorly understood. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to reveal aspects of the contribution of the Saccharomyces cerevisiae DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM). A tel1Δ mutant has globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tel1 mutation similarly increases Spo11-oligonucleotide levels but mutating known Tel1 phosphotargets on Hop1 and Rec114 does not, implicating Tel1 kinase activity and clarifying roles of Tel1 phosphorylation substrates. Deep sequencing of Spo11 oligonucleotides demonstrates that Tel1 shapes the genome-wide DSB landscape in unexpected ways. Early in meiosis, Tel1 absence causes widespread changes in DSB distributions across large chromosomal domains. Many of these changes are erased as meiosis proceeds, however, illustrating homeostatic behavior of DSB regulatory systems. We further find that effects of Tel1 are distinct but partially overlapping with previously described contributions of the recombination regulator Cst9 (also known as Zip3). Finally, we provide evidence indicating that Tel1-dependent DSB interference influences the population-average DSB landscape but also demonstrate that locally inhibitory effects of an artificial hotspot insertion can be both Tel1-independent and chromosomal context-dependent. Our findings delineate Tel1 roles in regulating number and location of DSBs and illuminate the complex interplay between Tel1 and other pathways for DSB control.
DNA double-strand breaks that initiate meiotic recombination are exonucleolytically processed. This 5′→3′ resection is a central, conserved feature of recombination but remains poorly understood. To ...address this lack, we mapped resection endpoints genome-wide at high resolution in Saccharomyces cerevisiae. Full-length resection requires Exo1 exonuclease and the DSB-responsive kinase Tel1, but not Sgs1 helicase. Tel1 also promotes efficient and timely resection initiation. Resection endpoints display pronounced heterogeneity between genomic loci that reflects a tendency for nucleosomes to block Exo1, yet Exo1 also appears to digest chromatin with high processivity and at rates similar to naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a defining feature of the meiotic resection landscape.
The number of DNA double-strand breaks (DSBs) initiating meiotic recombination is elevated in
mutants that are globally defective in forming crossovers and synaptonemal complex (SC), a protein ...scaffold juxtaposing homologous chromosomes. These mutants thus appear to lack a negative feedback loop that inhibits DSB formation when homologs engage one another. This feedback is predicted to be chromosome autonomous, but this has not been tested. Moreover, what chromosomal process is recognized as "homolog engagement" remains unclear. To address these questions, we evaluated effects of homolog engagement defects restricted to small portions of the genome using karyotypically abnormal yeast strains with a homeologous chromosome V pair, monosomic V, or trisomy XV. We found that homolog engagement-defective chromosomes incurred more DSBs, concomitant with prolonged retention of the DSB-promoting protein Rec114, while the rest of the genome remained unaffected. SC-deficient, crossover-proficient mutants
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experienced increased DSB numbers diagnostic of homolog engagement defects. These findings support the hypothesis that SC formation provokes DSB protein dissociation, leading in turn to loss of a DSB competent state. Our findings show that DSB number is regulated in a chromosome-autonomous fashion and provide insight into how homeostatic DSB controls respond to aneuploidy during meiosis.
Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by Spo11. In Saccharomyces cerevisiae, many DSBs occur in "hotspots" coinciding with nucleosome-depleted gene promoters. ...Transcription factors (TFs) stimulate DSB formation in some hotspots, but TF roles are complex and variable between locations. Until now, available data for TF effects on global DSB patterns were of low spatial resolution and confined to a single TF. Here, we examine at high resolution the contributions of two TFs to genome-wide DSB distributions: Bas1, which was known to regulate DSB activity at some loci, and Ino4, for which some binding sites were known to be within strong DSB hotspots. We examined fine-scale DSB distributions in TF mutant strains by deep sequencing oligonucleotides that remain covalently bound to Spo11 as a byproduct of DSB formation, mapped Bas1 and Ino4 binding sites in meiotic cells, evaluated chromatin structure around DSB hotspots, and measured changes in global messenger RNA levels. Our findings show that binding of these TFs has essentially no predictive power for DSB hotspot activity and definitively support the hypothesis that TF control of DSB numbers is context dependent and frequently indirect. TFs often affected the fine-scale distributions of DSBs within hotspots, and when seen, these effects paralleled effects on local chromatin structure. In contrast, changes in DSB frequencies in hotspots did not correlate with quantitative measures of chromatin accessibility, histone H3 lysine 4 trimethylation, or transcript levels. We also ruled out hotspot competition as a major source of indirect TF effects on DSB distributions. Thus, counter to prevailing models, roles of these TFs on DSB hotspot strength cannot be simply explained via chromatin "openness," histone modification, or compensatory interactions between adjacent hotspots.
Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ...ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of crossovers. Taken together, our results offer insights into the structure-function relationships of the MutLγ complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery.
Meiotic recombination, which promotes proper homologous chromosome segregation at the first meiotic division, normally occurs between allelic sequences on homologues. However, recombination can also ...take place between non-allelic DNA segments that share high sequence identity. Such non-allelic homologous recombination (NAHR) can markedly alter genome architecture during gametogenesis by generating chromosomal rearrangements. Indeed, NAHR-mediated deletions, duplications, inversions and other alterations have been implicated in numerous human genetic disorders. Studies in yeast have provided insights into the molecular mechanisms of meiotic NAHR as well as the cellular strategies that limit it.
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