Factors That Affect the Location and Frequency of Meiosis-Induced Double-Strand Breaks in Saccharomyces Cerevisiae

Double-strand DNA breaks (DSBs) initiate meiotic recombination in Saccharomyces cerevisiae. DSBs occur at sites that are hypersensitive in nuclease digests of chromatin, suggesting a role for chromatin structure in determining DSB location. We show here that the frequency of DSBs at a site is not determined simply by DNA sequence or by features of chromatin structure. An arg4-containing plasmid was inserted at several different locations in the yeast genome. Meiosis-induced DSBs occurred at similar sites in pBR322-derived portions of the construct at all insert loci, and the frequency of these breaks varied in a manner that mirrored the frequency of meiotic recombination in the arg4 portion of the insert. However, DSBs did not occur in the insert-borne arg4 gene at a site that is frequently broken at the normal ARG4 locus, even though the insert-borne arg4 gene and the normal ARG4 locus displayed similar DNase I hypersensitivity patterns. Deletions that removed active DSB sites from an insert at HIS4 restored breaks to the insert-borne arg4 gene and to a DSB site in flanking chromosomal sequences. We conclude that the frequency of DSB at a site can be affected by sequences several thousands nucleotides away and suggest that this is because of competition between DSB sites for locally limited factors.     

Changes in chromatin structure at recombination initiation sites during yeast meiosis.

Transient double-strand breaks (DSBs) occur during Saccharomyces cerevisiae meiosis at recombination hot spots and are thought to initiate most, if not all, homologous recombination between chromosomes. To uncover the regulatory mechanisms active in DSB formation, we have monitored the change in local chromatin structure at the ARG4 and CYS3 recombination hot spots over the course of meiosis. Micrococcal nuclease (MNase) digestion of isolated meiotic chromatin followed by indirect end-labeling revealed that the DSB sites in both loci are hypersensitive to MNase and that their sensitivity increases 2- to 4-fold prior to the appearance of meiotic DSBs and recombination products. Other sensitive sites are not significantly altered. The study of hyper- and hypo-recombinogenic constructs at the ARG4 locus, also revealed that the MNase sensitivity at the DSB site correlates with both the extent of DSBs and the rate of gene conversion. These results suggest that the local chromatin structure and its modification in early meiosis play an important role in the positioning and frequency of meiotic DSBs, leading to meiotic recombination.     

Correlation between premeiotic DNA replication and chromatin transition at yeast recombination initiation sites

The DNA double-strand breaks (DSBs) that initiate meiotic recombination in Saccharomyces cerevisiae are preceded first by DNA replication and then by a chromatin transition at DSB sites. This chromatin transition, detected as a quantitative increase in micrococcal nuclease (MNase) sensitivity, occurs specifically at DSB sites and not at other MNase-sensitive sites. Replication and DSB formation are directly linked: breaks do not form if replication is blocked, and delaying replication of a region also delays DSB formation in that region. We report here experiments that examine the relationship between replication, the DSB-specific chromatin transition and DSB formation. Deleting replication origins (and thus delaying replication) on the left arm of one of the two parental chromosomes III affects DSBs specifically on that replication-delayed arm and not those on the normally replicating arm. Thus, replication timing determines DSB timing in cis. Delaying replication on the left arm of chromosome III also delays the chromatin transition at DSB sites on that arm but not on the normally replicating right arm. Since the chromatin transition precedes DSB formation and requires the function of many genes necessary for DSB formation, these results suggest that initial events for DSB formation in chromatin are coupled with premeiotic DNA replication.     

DNA motif associated with meiotic double-strand break regions in Saccharomyces cerevisiae

Meiotic recombination in yeast is initiated by DNA double-strand breaks (DSBs) that occur at preferred sites, distributed along the chromosomes. These DSB sites undergo changes in chromatin structure early in meiosis, but their common features at the level of DNA sequence have not been defined until now. Alignment of 1 kb sequences flanking six well-mapped DSBs has allowed us to define a flexible sequence motif, the CoHR profile, which predicts the great majority of meiotic DSB locations. The 50 bp profile contains a poly(A) tract in its centre and may have several gaps of unrelated sequences over a total length of up to 250 bp. The major exceptions to the correlation between CoHRs and preferred DSB sites are at telomeric regions, where DSBs do not occur. The CoHR sequence may provide the basis for understanding meiosis-induced chromatin changes that enable DSBs to occur at defined chromosomal sites.     

Use of a Recombination Reporter Insert To Define Meiotic Recombination Domains on Chromosome III of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by DNA double-strand breaks (DSBs). DSBs usually occur in intergenic regions that display nuclease hypersensitivity in digests of chromatin. DSBs are distributed nonuniformly across chromosomes; on chromosome III, DSBs are concentrated in two "hot" regions, one in each chromosome arm. DSBs occur rarely in regions within about 40 kb of each telomere and in an 80-kb region in the center of the chromosome, just to the right of the centromere. We used recombination reporter inserts containing arg4 mutant alleles to show that the "cold" properties of the central DSB-deficient region are imposed on DNA inserted in the region. Cold region inserts display DSB and recombination frequencies that are substantially less than those seen with similar inserts in flanking hot regions. This occurs without apparent change in chromatin structure, as the same pattern and level of DNase I hypersensitivity is seen in chromatin of hot and cold region inserts. These data are consistent with the suggestion that features of higher-order chromosome structure or chromosome dynamics act in a target sequence-independent manner to control where recombination events initiate during meiosis.     

Long palindromic sequences induce double-strand breaks during meiosis in yeast

Inverted-repeated or palindromic sequences have been found to occur in both prokaryotic and eukaryotic genomes. Such repeated sequences are usually short and present at several functionally important regions in the genome. However, long palindromic sequences are rare and are a major source of genomic instability. The palindrome-mediated genomic instability is believed to be due to cruciform or hairpin formation and subsequent cleavage of this structure by structure-specific nucleases. Here we present both genetic and physical evidence that long palindromic sequences (>50 bp) generate double-strand breaks (DSBs) at a high frequency during meiosis in the yeast Saccharomyces cerevisiae. The palindrome-mediated DSB formation depends on the primary sequence of the inverted repeat and the location and length of the repeated units. The DSB formation at the palindrome requires all of the gene products that are known to be responsible for DSB formation at the normal meiosis-specific sites. Since DSBs are initiators of nearly all meiotic recombination events, most of the palindrome-induced breaks appear to be repaired by homologous recombination. Our results suggest that short palindromic sequences are highly stable in vivo. In contrast, long palindromic sequences make the genome unstable by inducing DSBs and such sequences are usually removed from the genome by homologous recombination events.     

Role of the Saccharomyces cerevisiae Rad53 checkpoint kinase in signaling double-strand breaks during the meiotic cell cycle

DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase, whereas the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed meiotic DSBs. Here we provide evidence that exogenous DSBs lead to Rad53 phosphorylation during the meiotic cell cycle, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.     

Targeted stimulation of meiotic recombination

Meiotic recombination in Saccharomyces cerevisiae is initiated by programmed DNA double-strand breaks (DSBs), a process that requires the Spo11 protein. DSBs usually occur in intergenic regions that display open chromatin accessibility, but other determinants that control their frequencies and non-random chromosomal distribution remain obscure. We report that a Spo11 construct bearing the Gal4 DNA binding domain not only rescues spo11Delta spore inviability and catalyzes DSB formation at natural sites but also strongly stimulates DSB formation near Gal4 binding sites. At GAL2, a naturally DSB-cold locus, Gal4BD-Spo11 creates a recombinational hotspot that depends on all the other DSB gene functions, showing that the targeting of Spo11 to a specific site is sufficient to stimulate meiotic recombination that is under normal physiological control.     

The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae.

Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae occurs by localized DNA double-strand breaks (DSBs) at several locations in the genome, corresponding to hot spots for meiotic gene conversion and crossing over. The meiotic DSBs occur in regions of chromatin that are hypersensitive to nucleases. To gain insight into the molecular mechanism involved in the formation of these DSBs, we have determined their positions at the nucleotide level at the CYS3 hot spot of gene conversion on chromosome I. We found four major new features of these DSBs: (i) sites of DSBs are multiple with varying intensities and spacing within the promoter region of the CYS3 gene; (ii) no consensus sequence can be found at these sites, indicating that the activity involved in DSB formation has little or no sequence specificity; (iii) the breaks are generated by blunt cleavages; and (iv) the 5' ends are modified in rad50S mutant strains, where the processing of these ends is known to be prevented. We present a model for the initiation of meiotic recombination taking into account the implications of these results.     

Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination

Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in?a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.     

C. elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination

Initiation of meiotic recombination by double-strand breaks (DSBs) must occur in a controlled fashion to avoid jeopardizing genome integrity. Here, we identify chromatin-associated protein HIM-17 as a link between chromatin state and DSB formation during C. elegans meiosis. Dependencies of several meiotic prophase events on HIM-17 parallel those seen for DSB-generating enzyme SPO-11: HIM-17 is essential for DSB formation but dispensable for homolog synapsis. Crossovers and chiasmata are eliminated in him-17 null mutants but are restored by artificially induced DSBs, indicating that all components required to convert DSBs into chiasmata are present. Unlike SPO-11, HIM-17 is also required for proper accumulation of histone H3 methylation at lysine 9 on meiotic prophase chromosomes. HIM-17 shares structural features with three proteins that interact genetically with LIN-35/Rb, a known component of chromatin-modifying complexes. Furthermore, DSB levels and incidence of chiasmata can be modulated by loss of LIN-35/Rb. These and other data suggest that chromatin state governs the timing of DSB competence.     

Sequence non-specific double-strand breaks and interhomolog interactions prior to double-strand break formation at a meiotic recombination hot spot in yeast.

The HIS4LEU2 meiotic recombination hot spot specifies two double-strand break (DSB) sites, I and II. Results presented demonstrate that DSBs at site I occur at many positions throughout a region of approximately 150 bp; we infer that breaks occur in a sequence non-specific fashion. Single-strand nicks at sites I and II are not detectable. Analysis of the effects of a 36 bp linker insertion at site I reveals the existence of communication along and between homologs prior to DSB formation. In cis, the insertion allele causes an increase in DSBs at site I but a decrease in DSBs at site II. In trans, two effects are observed. One effect likely reflects very early pre-DSB interhomolog interactions; the second is suggestive of a later, more intimate interaction in which sites I and II on the two homologs all compete for DSBs. The existence of interhomolog interactions in early meiotic prophase can explain how the sites of crossovers come to lie between the homolog axes at pachytene.     

RSC mobilizes nucleosomes to improve accessibility of repair machinery to the damaged chromatin

Repair of DNA double-strand breaks (DSBs) protects cells and organisms, as well as their genome integrity. Since DSB repair occurs in the context of chromatin, chromatin must be modified to prevent it from inhibiting DSB repair. Evidence supports the role of histone modifications and ATP-dependent chromatin remodeling in repair and signaling of chromosome DSBs. The key questions are, then, what the nature of chromatin altered by DSBs is and how remodeling of chromatin facilitates DSB repair. Here we report a chromatin alteration caused by a single HO endonuclease-generated DSB at the Saccharomyces cerevisiae MAT locus. The break induces rapid nucleosome migration to form histone-free DNA of a few hundred base pairs immediately adjacent to the break. The DSB-induced nucleosome repositioning appears independent of end processing, since it still occurs when the 5'-to-3' degradation of the DNA end is markedly reduced. The tetracycline-controlled depletion of Sth1, the ATPase of RSC, or deletion of RSC2 severely reduces chromatin remodeling and loading of Mre11 and Yku proteins at the DSB. Depletion of Sth1 also reduces phosphorylation of H2A, processing, and joining of DSBs. We propose that RSC-mediated chromatin remodeling at the DSB prepares chromatin to allow repair machinery to access the break and is vital for efficient DSB repair.     

Both conserved and non-conserved regions of Spo11 are essential for meiotic recombination initiation in yeast

DNA double-strand breaks (DSBs) are the initiators of most meiotic recombination events. In Saccharomyces cerevisiae, at least ten genes are necessary for meiotic DSB formation. However, the molecular roles of these proteins are not clearly understood. The meiosis-specific Spo11 protein, which shows sequence similarity with a subunit of an archaeal topoisomerase, is believed to catalyze the meiotic DSB formation. Spo11 is also required for induction of meiotic DSBs at long inverted repeats and at large trinucleotide repeat tracts. Here we report the isolation and characterization of temperature-sensitive spo11-mutant alleles to better understand how Spo11 functions, and how meiotic DSBs are generated at various recombination hotspots. Analysis of mutation sites of isolated spo11-mutant alleles indicated that both N-terminal and C-terminal non-conserved residues of Spo11 are essential for the protein’s function, possibly for interaction with other meiotic DSB enzymes. Several of the mutation sites within the conserved region are predicted to lie on the surface of the protein, suggesting that this region is required for activation of the meiotic initiation complex via protein-protein interaction. In addition to the conditional mutants, we isolated partially recombination-defective mutants; analysis of one of these mutants indicated that Ski8, as observed previously, interacts with Spo11 via the latter’s C-terminal residues.     

Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair

We developed a novel system to create DNA double-strand breaks (DSBs) at defined endogenous sites in the human genome, and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation (ChIP). The detection of human ATM protein at site-specific DSBs required functional NBS1 protein, ATM kinase activity and ATM autophosphorylation on Ser 1981. DSB formation led to the localized disruption of nucleosomes, a process that depended on both functional NBS1 and ATM. These two proteins were also required for efficient recruitment of the repair cofactor XRCC4 to DSBs, and for efficient DSB repair. These results demonstrate the functional importance of ATM kinase activity and phosphorylation in the response to DSBs, and support a model in which ordered chromatin structure changes that occur after DNA breakage depend on functional NBS1 and ATM, and facilitate DNA DSB repair.     

The C. elegans DSB-2 Protein Reveals a Regulatory Network that Controls Competence for Meiotic DSB Formation and Promotes Crossover Assurance

For most organisms, chromosome segregation during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs) and repair of a subset of these DSBs as inter-homolog crossovers (COs). However, timing and levels of DSB formation must be tightly controlled to avoid jeopardizing genome integrity. Here we identify the DSB-2 protein, which is required for efficient DSB formation during C. elegans meiosis but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of DSB formation, and its disappearance coincides with a decline in RAD-51 foci marking early recombination intermediates and precedes appearance of COSA-1 foci marking CO-designated sites. These and other data suggest that DSB-2 and its paralog DSB-1 promote competence for DSB formation. Further, immunofluorescence analyses of wild-type gonads and various meiotic mutants reveal that association of DSB-2 with chromatin is coordinated with multiple distinct aspects of the meiotic program, including the phosphorylation state of nuclear envelope protein SUN-1 and dependence on RAD-50 to load the RAD-51 recombinase at DSB sites. Moreover, association of DSB-2 with chromatin is prolonged in mutants impaired for either DSB formation or formation of downstream CO intermediates. These and other data suggest that association of DSB-2 with chromatin is an indicator of competence for DSB formation, and that cells respond to a deficit of CO-competent recombination intermediates by prolonging the DSB-competent state. In the context of this model, we propose that formation of sufficient CO-competent intermediates engages a negative feedback response that leads to cessation of DSB formation as part of a major coordinated transition in meiotic prophase progression. The proposed negative feedback regulation of DSB formation simultaneously (1) ensures that sufficient DSBs are made to guarantee CO formation and (2) prevents excessive DSB levels that could have deleterious effects.     

mei-P22 encodes a chromosome-associated protein required for the initiation of meiotic recombination in Drosophila melanogaster

Double-strand breaks (DSB) initiate meiotic recombination in a variety of organisms. Here we present genetic evidence that the mei-P22 gene is required for the induction of DSBs during meiotic prophase in Drosophila females. Strong mei-P22 mutations eliminate meiotic crossing over and suppress the sterility of DSB repair-defective mutants. Interestingly, crossing over in mei-P22 mutants can be restored to almost 50% of wild-type by X irradiation. In addition, an antibody-based assay was used to demonstrate that DSBs are not formed in mei-P22 mutants. This array of phenotypes is identical to that of mei-W68 mutants; mei-W68 encodes the Drosophila Spo11 homolog that is proposed to be an enzyme required for DSB formation. Consistent with a direct role in DSB formation, mei-P22 encodes a basic 35.7-kD protein, which, when examined by immunofluorescence, localizes to foci on meiotic chromosomes. MEI-P22 foci appear transiently in early meiotic prophase, which is when meiotic recombination is believed to initiate. By using an antibody to C(3)G as a marker for synaptonemal complex (SC) formation, we observed that SC is present before MEI-P22 associates with the chromosomes, thus providing direct evidence that the development of SC precedes the initiation of meiotic recombination. Similarly, we found that MEI-P22 foci did not appear in a c(3)G mutant in which SC does not form, suggesting that DSB formation is dependent on SC formation in Drosophila. We propose that MEI-P22 interacts with meiosis-specific chromosome proteins to facilitate DSB creation by MEI-W68.     

HTP-3 links DSB formation with homolog pairing and crossing over during C. elegans meiosis

Repair of the programmed meiotic double-strand breaks (DSBs) that initiate recombination must be coordinated with homolog pairing to generate crossovers capable of directing chromosome segregation. Chromosome pairing and synapsis proceed independently of recombination in worms and flies, suggesting a paradoxical lack of coregulation. Here, we find that the meiotic axis component HTP-3 links DSB formation with homolog pairing and synapsis. HTP-3 forms complexes with the DSB repair components MRE-11/RAD-50 and the meiosis-specific axis component HIM-3. Loss of htp-3 or mre-11 recapitulates meiotic phenotypes consistent with a failure to generate DSBs, suggesting that HTP-3 associates with MRE-11/RAD-50 in a complex required for meiotic DSB formation. Loss of HTP-3 eliminates HIM-3 localization to axes and HIM-3-dependent homolog alignment, synapsis, and crossing over. Our study reveals a mechanism for coupling meiotic DSB formation with homolog pairing through the essential participation of an axis component with complexes mediating both processes.