DNA cytosine methyltransferases differentially regulate genome-wide hypermutation and interhomolog recombination in Trichoderma reesei meiosis.

Trichoderma reesei is an economically important enzyme producer with several unique meiotic features. spo11, the initiator of meiotic double-strand breaks (DSBs) in most sexual eukaryotes, is dispensable for T. reesei meiosis. T. reesei lacks the meiosis-specific recombinase Dmc1. Rad51 and Sae2, the activator of the Mre11 endonuclease complex, promote DSB repair and chromosome synapsis in wild-type and spo11Δ meiosis. DNA methyltransferases (DNMTs) perform multiple tasks in meiosis. Three DNMT genes (rid1, dim2 and dimX) differentially regulate genome-wide cytosine methylation and C:G-to-T:A hypermutations in different chromosomal regions. We have identified two types of DSBs: type I DSBs require spo11 or rid1 for initiation, whereas type II DSBs do not rely on spo11 and rid1 for initiation. rid1 (but not dim2) is essential for Rad51-mediated DSB repair and normal meiosis. rid1 and rad51 exhibit a locus heterogeneity (LH) relationship, in which LH-associated proteins often regulate interconnectivity in protein interaction networks. This LH relationship can be suppressed by deleting dim2 in a haploid rid1Δ (but not rad51Δ) parental strain, indicating that dim2 and rid1 share a redundant function that acts earlier than rad51 during early meiosis. In conclusion, our studies provide the first evidence of the involvement of DNMTs during meiotic initiation and recombination.

Noncanonical usage of stop codons in ciliates expands proteins with structurally flexible Q-rich motifs.

Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

Trichoderma reesei Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences.

Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.

Budding yeast Rad51: a paradigm for how phosphorylation and intrinsic structural disorder regulate homologous recombination and protein homeostasis.

The RecA-family recombinase Rad51 is the central player in homologous recombination (HR), the faithful pathway for repairing DNA double-strand breaks (DSBs) during both mitosis and meiosis. The behavior of Rad51 protein in vivo is fine-tuned via posttranslational modifications conducted by multiple protein kinases in response to cell cycle cues and DNA lesions. Unrepaired DSBs and ssDNA also activate Mec1ATR and Tel1ATM family kinases to initiate the DNA damage response (DDR) that safeguards genomic integrity. Defects in HR and DDR trigger genome instability and result in cancer predisposition, infertility, developmental defects, neurological diseases or premature aging. Intriguingly, yeast Mec1ATR- and Tel1ATM-dependent phosphorylation promotes Rad51 protein stability during DDR, revealing how Mec1ATR can alleviate proteotoxic stress. Moreover, Mec1ATR- and Tel1ATM-dependent phosphorylation also occurs on DDR-unrelated proteins, suggesting that Mec1ATR and Tel1ATM have a DDR-independent function in protein homeostasis. In this minireview, we first describe how human and budding yeast Rad51 are phosphorylated by multiple protein kinases at different positions to promote homology-directed DNA repair and recombination (HDRR). Then, we discuss recent findings showing that intrinsic structural disorder and Mec1ATR/Tel1ATM-dependent phosphorylation are coordinated in yeast Rad51 to regulate both HR and protein homeostasis.

Dual roles of yeast Rad51 N-terminal domain in repairing DNA double-strand breaks.

Highly toxic DNA double-strand breaks (DSBs) readily trigger the DNA damage response (DDR) in cells, which delays cell cycle progression to ensure proper DSB repair. In Saccharomyces cerevisiae, mitotic S phase (20-30 min) is lengthened upon DNA damage. During meiosis, Spo11-induced DSB onset and repair lasts up to 5 h. We report that the NH2-terminal domain (NTD; residues 1-66) of Rad51 has dual functions for repairing DSBs during vegetative growth and meiosis. Firstly, Rad51-NTD exhibits autonomous expression-enhancing activity for high-level production of native Rad51 and when fused to exogenous ß-galactosidase in vivo. Secondly, Rad51-NTD is an S/T-Q cluster domain (SCD) harboring three putative Mec1/Tel1 target sites. Mec1/Tel1-dependent phosphorylation antagonizes the proteasomal degradation pathway, increasing the half-life of Rad51 from ∼30 min to ≥180 min. Our results evidence a direct link between homologous recombination and DDR modulated by Rad51 homeostasis.

RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway.

Members of the ribonuclease A (RNase A) superfamily regulate various physiological processes. RNase A, the best-studied member of the RNase A superfamily, is widely expressed in different tissues, including brains. We unexpectedly found that RNase A can trigger proliferation of neuronal progenitor cells (NPC) both in vitro and in vivo. RNase A treatment induced cell proliferation in dissociated neuronal cultures and increased cell mass in neurosphere cultures. BrdU (5-Bromo-2'-Deoxyuridine) labeling confirmed the effect of RNase A on cell proliferation. Those dividing cells were Nestin- and SOX2-positive, suggesting that RNase A triggers NPC proliferation. The proliferation inhibitor Ara-C completely suppressed the effect of RNase A on NPC counts, further supporting that RNase A increases NPC number mainly by promoting proliferation. Moreover, we found that RNase A treatment increased ERK phosphorylation and blockade of the ERK pathway inhibited the effect of RNase A on NPC proliferation. Intracerebroventricular injection of RNase A into mouse brain increased the population of 5-ethynyl-2'-deoxyuridine (EdU) or BrdU-labeled cells in the subventricular zone. Those RNase A-induced NPCs were able to migrate into other brain areas, including hippocampus, amygdala, cortex, striatum, and thalamus. In conclusion, our study shows that RNase A promotes proliferation of NPCs via an ERK-dependent pathway and further diversifies the physiological functions of the RNase A family.

S. cerevisiae Mre11 recruits conjugated SUMO moieties to facilitate the assembly and function of the Mre11-Rad50-Xrs2 complex.

Double-strand breaks (DSBs) in chromosomes are the most challenging type of DNA damage. The yeast and mammalian Mre11-Rad50-Xrs2/Nbs1 (MRX/N)-Sae2/Ctp1 complex catalyzes the resection of DSBs induced by secondary structures, chemical adducts or covalently-attached proteins. MRX/N also initiates two parallel DNA damage responses-checkpoint phosphorylation and global SUMOylation-to boost a cell's ability to repair DSBs. However, the molecular mechanism of this SUMO-mediated response is not completely known. In this study, we report that Saccharomyces cerevisiae Mre11 can non-covalently recruit the conjugated SUMO moieties, particularly the poly-SUMO chain. Mre11 has two evolutionarily-conserved SUMO-interacting motifs, Mre11(SIM1) and Mre11(SIM2), which reside on the outermost surface of Mre11. Mre11(SIM1) is indispensable for MRX assembly. Mre11(SIM2) non-covalently links MRX with the SUMO enzymes (E2/Ubc9 and E3/Siz2) to promote global SUMOylation of DNA repair proteins. Mre11(SIM2) acts independently of checkpoint phosphorylation. During meiosis, the mre11(SIM2) mutant, as for mre11S, rad50S and sae2Δ, allows initiation but not processing of Spo11-induced DSBs. Using MRX and DSB repair as a model, our work reveals a general principle in which the conjugated SUMO moieties non-covalently facilitate the assembly and functions of multi-subunit protein complexes.

Pch2 prevents Mec1/Tel1-mediated Hop1 phosphorylation occurring independently of Red1 in budding yeast meiosis.

A prominent feature of meiosis in most sexually reproducing organisms is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks are repaired using intact homologous non-sister chromatids rather than sister chromatids. Budding yeast DNA damage checkpoint kinases Mec1 and Tel1 act together with the axial element protein Red1 to promote interhomolog recombination by phosphorylating another axial element protein Hop1. Mec1 and Tel1 also phosphorylate γH2A and the synaptonemal complex protein Zip1 independently of Red1 to facilitate premeiotic DNA replication and to destabilize homology-independent centromere pairing, respectively. It has been unclear why Hop1 phosphorylation is Red1-dependent. Here, we report that the pachytene checkpoint protein 2 (Pch2) specifically prevents Red1-independent Hop1 phosphorylation. Our findings reveal a new function for Pch2 in linking two axial element proteins Red1 and Hop1 thus coordinating their effects in meiotic recombination and the checkpoint network.

Interorganelle interactions and inheritance patterns of nuclei and vacuoles in budding yeast meiosis.

Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.

Three distinct modes of Mec1/ATR and Tel1/ATM activation illustrate differential checkpoint targeting during budding yeast early meiosis.

Recombination and synapsis of homologous chromosomes are hallmarks of meiosis in many organisms. Meiotic recombination is initiated by Spo11-induced DNA double-strand breaks (DSBs), whereas chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). Previously, we proposed that budding yeast SC is assembled via noncovalent interactions between the axial SC protein Red1, SUMO chains or conjugates, and the central SC protein Zip1. Incomplete synapsis and unrepaired DNA are monitored by Mec1/Tel1-dependent checkpoint responses that prevent exit from the pachytene stage. Here, our results distinguished three distinct modes of Mec1/Tec1 activation during early meiosis that led to phosphorylation of three targets, histone H2A at S129 (γH2A), Hop1, and Zip1, which are involved, respectively, in DNA replication, the interhomolog recombination and chromosome synapsis checkpoint, and destabilization of homology-independent centromere pairing. γH2A phosphorylation is Red1 independent and occurs prior to Spo11-induced DSBs. DSB- and Red1-dependent Hop1 phosphorylation is activated via interaction of the Red1-SUMO chain/conjugate ensemble with the Ddc1-Rad17-Mec3 (9-1-1) checkpoint complex and the Mre11-Rad50-Xrs2 complex. During SC assembly, Zip1 outcompetes 9-1-1 from the Red1-SUMO chain ensemble to attenuate Hop1 phosphorylation. In contrast, chromosome synapsis cannot attenuate DSB-dependent and Red1-independent Zip1 phosphorylation. These results reveal how DNA replication, DSB repair, and chromosome synapsis are differentially monitored by the meiotic checkpoint network.

Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis.

Red1, Hop1 and Mek1 are three yeast meiosis-specific chromosomal proteins that uphold the interhomolog (IH) bias of meiotic recombination. Mek1 is also an effector protein kinase in a checkpoint that responds to aberrant DNA and/or axis structure. The activation of Mek1 requires Red1-dependent Hop1-Thr(T)318 phosphorylation, which is mediated by Mec1 and Tel1, the yeast homologs of the mammalian DNA damage sensor kinases ATR and ATM. As the ectopic expression of Mek1-glutathione S-transferase (GST) was shown to promote IH recombination in the absence of Mec1/Tel1-dependent checkpoint function, it was proposed that Mek1 might play dual roles during meiosis by directly phosphorylating targets that are involved in the recombination checkpoint. Here, we report that Mek1 has a positive feedback activity in the stabilization of Mec1/Tel1-mediated Hop1-T318 phosphorylation against the dephosphorylation mediated by protein phosphatase 4. Our results also reveal that GST-Mek1 or Mek1-GST further increases Hop1-T318 phosphorylation. This positive feedback function of Mek1 is independent of Mek1's kinase activity, but dependent on Mek1's forkhead-associated (FHA) domain and its arginine 51 residue. Arginine 51 directly mediates the interaction of Mek1-FHA and phosphorylated Hop1-T318. We suggest that the Hop1-Mek1 interaction is similar to the Rad53-Dun1 signaling pathway, which is mediated through the interaction of phosphorylated Rad53 and Dun1-FHA.