The DNA Replication Factor RFC1 Is Required for Interference-Sensitive Meiotic Crossovers in Arabidopsis thaliana

During meiotic recombination, induced double-strand breaks (DSBs) are processed into crossovers (COs) and non-COs (NCO); the former are required for proper chromosome segregation and fertility. DNA synthesis is essential in current models of meiotic recombination pathways and includes only leading strand DNA synthesis, but few genes crucial for DNA synthesis have been tested genetically for their functions in meiosis. Furthermore, lagging strand synthesis has been assumed to be unnecessary. Here we show that the Arabidopsis thaliana DNA REPLICATION FACTOR C1 (RFC1) important for lagging strand synthesis is necessary for fertility, meiotic bivalent formation, and homolog segregation. Loss of meiotic RFC1 function caused abnormal meiotic chromosome association and other cytological defects; genetic analyses with other meiotic mutations indicate that RFC1 acts in the MSH4-dependent interference-sensitive pathway for CO formation. In a rfc1 mutant, residual pollen viability is MUS81-dependent and COs exhibit essentially no interference, indicating that these COs form via the MUS81-dependent interference-insensitive pathway. We hypothesize that lagging strand DNA synthesis is important for the formation of double Holliday junctions, but not alternative recombination intermediates. That RFC1 is found in divergent eukaryotes suggests a previously unrecognized and highly conserved role for DNA synthesis in discriminating between recombination pathways.     

Genetic Analysis of Baker's Yeast Msh4-Msh5 Reveals a Threshold Crossover Level for Meiotic Viability

During meiosis, the Msh4-Msh5 complex is thought to stabilize single-end invasion intermediates that form during early stages of recombination and subsequently bind to Holliday junctions to facilitate crossover formation. To analyze Msh4-Msh5 function, we mutagenized 57 residues in Saccharomyces cerevisiae Msh4 and Msh5 that are either conserved across all Msh4/5 family members or are specific to Msh4 and Msh5. The Msh5 subunit appeared more sensitive to mutagenesis. We identified msh4 and msh5 threshold (msh4/5-t) mutants that showed wild-type spore viability and crossover interference but displayed, compared to wild-type, up to a two-fold decrease in crossing over on large and medium sized chromosomes (XV, VII, VIII). Crossing over on a small chromosome, however, approached wild-type levels. The msh4/5-t mutants also displayed synaptonemal complex assembly defects. A triple mutant containing a msh4/5-t allele and mutations that decreased meiotic double-strand break levels (spo11-HA) and crossover interference (pch2Δ) showed synergistic defects in spore viability. Together these results indicate that the baker''s yeast meiotic cell does not require the ∼90 crossovers maintained by crossover homeostasis to form viable spores. They also show that Pch2-mediated crossover interference is important to maintain meiotic viability when crossovers become limiting.     

Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor,Is a Msh2-Msh3-stimulated Endonuclease

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.     

Epistatic interactions between four rad loci in yeast

Haploid yeast strains carrying mutations in two or more of four ad genes were contrusted by tetrad dissection, and the UV survival of these strains was measured. It was found that (with one exception) double mutant strains were not significantly more sensitive than the most sensitive single mutants, for strains involving mutant loci rad 1, rad 3 and rad 4. The exception was the double mutant rad 1–5 rad 4-4, but another double mutant involving different alleles of the the same loci did not show an enhanced UV sensitivity. Triple and quadruple mutants also failed to show a significantly increased UV sensitivity with respect to the single mutants. The results indicate that all these four mutant loci confer UV sensitivity by the same mechanism, and it is suggested that the wild-type alleles mediate excision-repair of UV-induced DNA lesions. Enhanced sensitivity of the genotype rad 1–5 rad 4-4 is attributed to leakiness of these alleles.     

Fanconi anemia ortholog FANCM regulates meiotic crossover distribution in plants

Meiotic recombination increases genetic diversity and manipulation of its frequency and distribution holds great promise in crop breeding. In Arabidopsis thaliana, FANCM (a homolog of mammalian Fanconi anemia complementation group M) suppresses recombination and its function seems conserved in other species including the rosids Brassica spp. and pea (Pisum sativum), and the monocot rice (Oryza sativa). To examine the role of FANCM during meiotic recombination in lettuce (Lactuca sativa, an asterid), we characterized the function of lettuce LsFANCM and found that it can functionally substitute for AtFANCM in transgenic Arabidopsis plants. Moreover, three independent CRISPR/Cas9-edited lettuce Lsfancm mutants showed reduced pollen viability and seed setting. Unexpectedly, analyses of chromosome behavior revealed that 77.8% of Lsfancm meiocytes exhibited univalents. The normal formation of double-strand breaks in DNA and the discontinuous assembly of synaptonemal complex in Lsfancm mutants supports the hypothesis that LsFANCM might be dispensable for the initiation of meiotic recombination but required for normal synapsis. Furthermore, the frequency of lettuce HEI10 (Human Enhancer of Invasion 10) foci, a marker for Class-I crossovers (COs), was similar between wild-type (WT) and Lsfancm. Strikingly, the distribution of LsHEI10 foci and chiasmata in Lsfancm meiotic chromosomes was markedly different from the WT. A similar alteration in the distribution of Class-I COs was also observed in the Arabidopsis Atfancm mutant. Taken together, these results demonstrate that FANCM is important for shaping the distribution of meiotic Class-I COs in plants, and reveal an evolutionarily divergent role for FANCM in meiotic bivalent formation between Arabidopsis and lettuce.

FANCM has a role in shaping meiotic crossover in plants, and its function in meiotic bivalent formation has diverged in Arabidopsis and lettuce.     

Reduced Crossover Interference and Increased ZMM-Independent Recombination in the Absence of Tel1/ATM

Meiotic recombination involves the repair of double-strand break (DSB) precursors as crossovers (COs) or noncrossovers (NCOs). The proper number and distribution of COs is critical for successful chromosome segregation and formation of viable gametes. In budding yeast the majority of COs occurs through a pathway dependent on the ZMM proteins (Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3), which form foci at CO-committed sites. Here we show that the DNA-damage-response kinase Tel1/ATM limits ZMM-independent recombination. By whole-genome mapping of recombination products, we find that lack of Tel1 results in higher recombination and reduced CO interference. Yet the number of Zip3 foci in tel1Δ cells is similar to wild type, and these foci show normal interference. Analysis of recombination in a tel1Δ zip3Δ double mutant indicates that COs are less dependent on Zip3 in the absence of Tel1. Together these results reveal that in the absence of Tel1, a significant proportion of COs occurs through a non-ZMM-dependent pathway, contributing to a CO landscape with poor interference. We also see a significant change in the distribution of all detectable recombination products in the absence of Tel1, Sgs1, Zip3, or Msh4, providing evidence for altered DSB distribution. These results support the previous finding that DSB interference depends on Tel1, and further suggest an additional level of DSB interference created through local repression of DSBs around CO-designated sites.     

The role of rice HEI10 in the formation of meiotic crossovers

HEI10 was first described in human as a RING domain-containing protein that regulates cell cycle and cell invasion. Mice HEI10(mei4) mutant displays no obvious defect other than meiotic failure from an absence of chiasmata. In this study, we characterize rice HEI10 by map-based cloning and explore its function during meiotic recombination. In the rice hei10 mutant, chiasma frequency is markedly reduced, and those remaining chiasmata exhibit a random distribution among cells, suggesting possible involvement of HEI10 in the formation of interference-sensitive crossovers (COs). However, mutation of HEI10 does not affect early recombination events and synaptonemal complex (SC) formation. HEI10 protein displays a highly dynamic localization on the meiotic chromosomes. It initially appears as distinct foci and co-localizes with MER3. Thereafter, HEI10 signals elongate along the chromosomes and finally restrict to prominent foci that specially localize to chiasma sites. The linear HEI10 signals always localize on ZEP1 signals, indicating that HEI10 extends along the chromosome in the wake of synapsis. Together our results suggest that HEI10 is the homolog of budding yeast Zip3 and Caenorhabditis elegans ZHP-3, and may specifically promote class I CO formation through modification of various meiotic components.     

Competing crossover pathways act during meiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the MSH4-MSH5, MLH1-MLH3, and MUS81-MMS4 complexes act to promote crossing over during meiosis. MSH4-MSH5, but not MUS81-MMS4, promotes crossovers that display interference. A role for MLH1-MLH3 in crossover control is less clear partly because mlh1Delta mutants retain crossover interference yet display a decrease in crossing over that is only slightly less severe than that seen in msh4Delta and msh5Delta mutants. We analyzed the effects of msh5Delta, mlh1Delta, and mms4Delta single, double, and triple mutants on meiotic crossing over at four consecutive genetic intervals on chromosome XV using newly developed computer software. mlh1Delta mms4Delta double mutants displayed the largest decrease in crossing over (13- to 15-fold) of all mutant combinations, yet these strains displayed relatively high spore viability (42%). In contrast, msh5Delta mms4Delta and msh5Delta mms4Delta mlh1Delta mutants displayed smaller decreases in crossing over (4- to 6-fold); however, spore viability (18-19%) was lower in these strains than in mlh1Delta mms4Delta strains. These data suggest that meiotic crossing over can occur in yeast through three distinct crossover pathways. In one pathway, MUS81-MMS4 promotes interference-independent crossing over; in a second pathway, both MSH4-MSH5 and MLH1-MLH3 promote interference-dependent crossovers. A third pathway, which appears to be repressed by MSH4-MSH5, yields deleterious crossovers.     

Regulation of Msh4-Msh5 association with meiotic chromosomes in budding yeast

In the baker’s yeast Saccharomyces cerevisiae, most of the meiotic crossovers are generated through a pathway involving the highly conserved mismatch repair related Msh4-Msh5 complex. To understand the role of Msh4-Msh5 in meiotic crossing over, we determined its genome wide in vivo binding sites in meiotic cells. We show that Msh5 specifically associates with DSB hotspots, chromosome axes, and centromeres on chromosomes. A basal level of Msh5 association with these chromosomal features is observed even in the absence of DSB formation (spo11Δ mutant) at the early stages of meiosis. But efficient binding to DSB hotspots and chromosome axes requires DSB formation and resection and is enhanced by double Holliday junction structures. Msh5 binding is also correlated to DSB frequency and enhanced on small chromosomes with higher DSB and crossover density. The axis protein Red1 is required for Msh5 association with the chromosome axes and DSB hotspots but not centromeres. Although binding sites of Msh5 and other pro-crossover factors like Zip3 show extensive overlap, Msh5 associates with centromeres independent of Zip3. These results on Msh5 localization in wild type and meiotic mutants have implications for how Msh4-Msh5 works with other pro-crossover factors to ensure crossover formation.     

Functional overlap between the structure-specific nucleases Yen1 and Mus81-Mms4 for DNA-damage repair in S. cerevisiae

In eukaryotic cells, multiple DNA repair mechanisms respond to a wide variety of DNA lesions. Homologous recombination-dependent repair provides a pathway for dealing with DNA double-strand breaks and replication fork demise. A key step in this process is the resolution of recombination intermediates such as Holliday junctions (HJs). Recently, nucleases from yeast (Yen1) and human cells (GEN1) were identified that can resolve HJ intermediates, in a manner analogous to the E. coli HJ resolvase RuvC. Here, we have analyzed the role of Yen1 in DNA repair in S. cerevisiae, and show that while yen1Δ mutants are repair-proficient, yen1Δ mus81Δ double mutants are exquisitely sensitive to a variety of DNA-damaging agents that disturb replication fork progression. This phenotype is dependent upon RAD52, indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81. After MMS treatment, yen1Δ mus81Δ double mutants arrest with a G2 DNA content and unsegregated chromosomes. These findings indicate that Yen1 can act upon recombination/repair intermediates that arise in MUS81-defective cells following replication fork damage.     

Partial suppression of the fission yeast rqh1(-) phenotype by expression of a bacterial Holliday junction resolvase

A key stage during homologous recombination is the processing of the Holliday junction, which determines the outcome of the recombination reaction. To dissect the pathways of Holliday junction processing in a eukaryote, we have targeted an Escherichia coli Holliday junction resolvase to the nuclei of fission yeast recombination-deficient mutants and analysed their phenotypes. The resolvase partially complements the UV and hydroxyurea hypersensitivity and associated aberrant mitoses of an rqh1(-) mutant. Rqh1 is a member of the RecQ subfamily of DNA helicases that control recombination particularly during S-phase. Significantly, overexpression of the resolvase in wild-type cells partly mimics the loss of viability, hyper-recombination and 'cut' phenotype of an rqh1(-) mutant. These results indicate that Holliday junctions form in wild-type cells that are normally removed in a non-recombinogenic way, possibly by Rqh1 catalysing their reverse branch migration. We propose that in the absence of Rqh1, replication fork arrest results in the accumulation of Holliday junctions, which can either impede sister chromatid segregation or lead to the formation of recombinants through Holliday junction resolution.     

Reduced meiotic crossovers and delayed prophase I progression in AtMLH3-deficient Arabidopsis

Characterization of AtMLH3, the Arabidopsis homologue of the prokaryotic MutL mismatch repair gene, reveals that it is expressed in reproductive tissue where it is required for normal levels of meiotic crossovers (COs). Immunocytological studies in an Atmlh3 mutant indicate that chromosome pairing and synapsis proceed with normal distribution of the early recombination pathway proteins. Localization of the MutS homologue AtMSH4 occurs, suggesting that double Holliday junctions (dHjs) are formed, but the MutL homologue AtMLH1, which forms a heterocomplex with AtMLH3, fails to localize normally. Loss of AtMLH3 results in an approximately 60% reduction in COs and is accompanied by a substantial delay of approximately 25 h in prophase I progression. Analysis of the chiasma distribution in Atmlh3 suggests that dHj resolution can occur, but in contrast to wild type where most or all dHjs are directed to form COs the outcome is biased in favour of a non-CO outcome by a ratio of around 2 to 1. The data are compatible with a model whereby the MutL complex imposes a dHj conformation that ensures CO formation.     

Meiotic recombination intermediates are resolved with minimal crossover formation during return-to-growth, an analogue of the mitotic cell cycle

Accurate segregation of homologous chromosomes of different parental origin (homologs) during the first division of meiosis (meiosis I) requires inter-homolog crossovers (COs). These are produced at the end of meiosis I prophase, when recombination intermediates that contain Holliday junctions (joint molecules, JMs) are resolved, predominantly as COs. JM resolution during the mitotic cell cycle is less well understood, mainly due to low levels of inter-homolog JMs. To compare JM resolution during meiosis and the mitotic cell cycle, we used a unique feature of Saccharomyces cerevisiae, return to growth (RTG), where cells undergoing meiosis can be returned to the mitotic cell cycle by a nutritional shift. By performing RTG with ndt80 mutants, which arrest in meiosis I prophase with high levels of interhomolog JMs, we could readily monitor JM resolution during the first cell division of RTG genetically and, for the first time, at the molecular level. In contrast to meiosis, where most JMs resolve as COs, most JMs were resolved during the first 1.5-2 hr after RTG without producing COs. Subsequent resolution of the remaining JMs produced COs, and this CO production required the Mus81/Mms4 structure-selective endonuclease. RTG in sgs1-ΔC795 mutants, which lack the helicase and Holliday junction-binding domains of this BLM homolog, led to a substantial delay in JM resolution; and subsequent JM resolution produced both COs and NCOs. Based on these findings, we suggest that most JMs are resolved during the mitotic cell cycle by dissolution, an Sgs1 helicase-dependent process that produces only NCOs. JMs that escape dissolution are mostly resolved by Mus81/Mms4-dependent cleavage that produces both COs and NCOs in a relatively unbiased manner. Thus, in contrast to meiosis, where JM resolution is heavily biased towards COs, JM resolution during RTG minimizes CO formation, thus maintaining genome integrity and minimizing loss of heterozygosity.     

'Saccharomyces cerevisiae MSH2/6 complex interacts with Holliday junctions and facilitates their cleavage by phage resolution enzymes

Genetic and biochemical studies have indicated that mismatch repair proteins can interact with recombination intermediates. In this study, gel shift assays and electron microscopic analysis were used to show that the Saccharomyces cerevisiae MSH2/6 complex binds to Holliday junctions and has an affinity and specificity for them that is at least as high as it has as for mispaired bases. Under equilibrium binding conditions, the MSH2/6 complex had a Kd of binding to Holliday junctions of 0.5 nM. The MSH2/6 complex enhanced the cleavage of Holliday junctions by T4 endonuclease VII and T7 endonuclease I. This is consistent with the view that the MSH2/6 complex can function in both mismatch repair and the resolution of recombination intermediates as predicted by genetic studies.     

The human Holliday junction resolvase GEN1 rescues the meiotic phenotype of a Schizosaccharomyces pombe mus81 mutant

A key step in meiotic recombination involves the nucleolytic resolution of Holliday junctions to generate crossovers. Although the enzyme that performs this function in human cells is presently unknown, recent studies led to the identification of the XPG-family endonuclease GEN1 that promotes Holliday junction resolution in vitro, suggesting that it may perform a related function in vivo. Here, we show that ectopic expression of GEN1 in fission yeast mus81Δ strains results in Holliday junction resolution and crossover formation during meiosis.     

Mph1 requires mismatch repair-independent and -dependent functions of MutSα to regulate crossover formation during homologous recombination repair

In budding yeast the DNA helicase Mph1 prevents genome rearrangements during ectopic homologous recombination (HR) by suppressing the formation of crossovers (COs). Here we show that during ectopic HR repair, the anti-CO function of Mph1 is intricately associated with the mismatch repair (MMR) factor, MutSα. In particular, during HR repair using a completely homologous substrate, we reveal an MMR-independent function of MutSα in generating COs that is specifically antagonized by Mph1, but not Sgs1. In contrast, both Mph1 and MutSα are required to efficiently suppress COs in the presence of a homeologous substrate. Mph1 acts redundantly with Sgs1 in this respect since mph1Δ sgs1Δ double mutant cells pheno-copy MutSα mutants and completely fail to discriminate homologous and homeologous sequences during HR repair. However, this defect of mph1Δ sgs1Δ cells is not due to an inability to carry out MMR but rather is accompanied by elevated levels of gene conversion (GC) and bi-directional GC tracts specifically in non-crossover products. Models describing how Mph1, MutSα and Sgs1 act in concert to suppress genome rearrangements during ectopic HR repair are discussed.     

Lack of mycothiol and ergothioneine induces different protective mechanisms in Mycobacterium smegmatis

Mycobacterium smegmatis contains the low molecular weight thiols, mycothiol (MSH) and ergothioneine (ESH). Examination of transposon mutants disrupted in mshC and egtA, involved in the biosynthesis of MSH and ESH respectively, demonstrated that both mutants were sensitive to oxidative, alkylating, and metal stress. However, the mshC mutant exhibited significantly more protein carbonylation and lipid peroxidation than wildtype, while the egtA mutant had less protein and lipid damage than wildtype. We further show that Ohr, KatN, and AhpC, involved in protection against oxidative stress, are upregulated in the egtA mutant. In the mshC mutant, an Usp and a putative thiol peroxidase are upregulated. In addition, mutants lacking MSH also contained higher levels of Coenzyme F420 as compared to wildtype and two Coenzyme F420 dependent enzymes were found to be upregulated. These results indicate that lack of MSH and ESH result in induction of different mechanisms for protecting against oxidative stress.     

ZHP-3 acts at crossovers to couple meiotic recombination with synaptonemal complex disassembly and bivalent formation in C. elegans

Crossover recombination and the formation of chiasmata normally ensure the proper segregation of homologous chromosomes during the first meiotic division. zhp-3, the Caenorhabditis elegans ortholog of the budding yeast ZIP3 gene, is required for crossover recombination. We show that ZHP-3 protein localization is highly dynamic. At a key transition point in meiotic prophase, the protein shifts from along the length of the synaptonemal complex (SC) to an asymmetric localization on the SC and eventually becomes restricted to foci that mark crossover recombination events. A zhp-3::gfp transgene partially complements a null mutation and reveals a separation of function; although the fusion protein can promote nearly wild-type levels of recombination, aneuploidy among the progeny is high, indicating defects in meiotic chromosome segregation. The structure of bivalents is perturbed in this mutant, suggesting that the chromosome segregation defect results from an inability to properly remodel chromosomes in response to crossovers. smo-1 mutants exhibit phenotypes similar to zhp-3::gfp mutants at higher temperatures, and smo-1; zhp-3::gfp double mutants exhibit more severe meiotic defects than either single mutant, consistent with a role for SUMO in the process of SC disassembly and bivalent differentiation. We propose that coordination of crossover recombination with SC disassembly and bivalent formation reflects a conserved role of Zip3/ZHP-3 in coupling recombination with SC morphogenesis.     

Differential effects of the mismatch repair genes MSH2 and MSH3 on homeologous recombination in Saccharomyces cerevisiae

The products of the yeast mismatch repair genes MSH2 and MSH3 participate in the inhibition of genetic recombination between homeologous (divergent) DNA sequences. In strains deficient for these genes, homeologous recombination rates between repeated elements are elevated due to the loss of this inhibition. In this study, the effects of these mutations were further analyzed by quantitation of mitotic homeologous recombinants as crossovers, gene conversions or exceptional events in wild-type, msh2, msh3 and msh2 msh3 mutant strains. When homeologous sequences were present as a direct repeat in one orientation, crossovers and gene conversions were elevated in msh2, msh3 and msh2 msh3 strains. The increases were greater in the msh2 msh3 double mutant than in either single mutant. When the order of the homeologous sequences was reversed, the msh2 mutation again yielded increased rates of crossovers and gene conversions. However, in an msh3 strain, gene conversions occurred at higher levels but interchromosomal crossovers were not increased and intrachromosomal crossovers were reduced relative to wild type. The msh2 msh3 double mutant behaved like the msh2 single mutant in this orientation. Control strains harboring homologous duplications were largely but not entirely unaffected in mutant strains, suggesting specificity for the mismatched intermediates of homeologous recombination. In all strains, very few (<10%) recombinants could be attributed to exceptional events. These results suggest that MSH2 and MSH3 can function differentially to control homeologous exchanges.     

The Arabidopsis thaliana PARTING DANCERS gene encoding a novel protein is required for normal meiotic homologous recombination

Recent studies of meiotic recombination in the budding yeast and the model plant Arabidopsis thaliana indicate that meiotic crossovers (COs) occur through two genetic pathways: the interference-sensitive pathway and the interference-insensitive pathway. However, few genes have been identified in either pathway. Here, we describe the identification of the PARTING DANCERS (PTD) gene, as a gene with an elevated expression level in meiocytes. Analysis of two independently generated transferred DNA insertional lines in PTD showed that the mutants had reduced fertility. Further cytological analysis of male meiosis in the ptd mutants revealed defects in meiosis, including reduced formation of chiasmata, the cytological appearance of COs. The residual chiasmata in the mutants were distributed randomly, indicating that the ptd mutants are defective for CO formation in the interference-sensitive pathway. In addition, transmission electron microscopic analysis of the mutants detected no obvious abnormality of synaptonemal complexes and apparently normal late recombination nodules at the pachytene stage, suggesting that the mutant's defects in bivalent formation were postsynaptic. Comparison to other genes with limited sequence similarity raises the possibility that PTD may present a previously unknown function conserved in divergent eukaryotic organisms.