Dna2 removes toxic ssDNA-RPA filaments generated from meiotic recombination-associated DNA synthesis.

During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strands can displace the parental strand to generate single-strand DNAs (ssDNAs). Many programmed DSBs and thus many ssDNAs occur during meiosis. However, it is unclear how these ssDNAs are removed for the complete repair of meiotic DSBs. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) results in an abundant accumulation of RPA and an expansion of RPA from DSBs to broader regions in Saccharomyces cerevisiae. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, Dna2 induction at pachytene is highly effective in removing accumulated RPA and restoring spore viability. Moreover, the depletion of Pif1, an activator of polymerase δ required for meiotic recombination-associated DNA synthesis, and Pif1 inhibitor Mlh2 decreases and increases RPA accumulation in dna2-md, respectively. In addition, blocking DNA synthesis during meiotic recombination dramatically decreases RPA accumulation in dna2-md. Together, our findings show that meiotic DSB repair requires Dna2 to remove ssDNA-RPA filaments generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.

Homologous chromosome pairing: The linchpin of accurate segregation in meiosis.

Meiosis is a specialized cell division that occurs in sexually reproducing organisms, generating haploid gametes containing half the chromosome number through two rounds of cell division. Homologous chromosomes pair and prepare for their proper segregation in subsequent divisions. How homologous chromosomes recognize each other and achieve pairing is an important question. Early studies showed that in most organisms, homologous pairing relies on homologous recombination. However, pairing mechanisms differ across species. Evidence indicates that chromosomes are dynamic and move during early meiotic stages, facilitating pairing. Recent studies in various model organisms suggest conserved mechanisms and key regulators of homologous chromosome pairing. This review summarizes these findings and compare similarities and differences in homologous chromosome pairing mechanisms across species.

Enzymatic Synthesis of Unprotected α,ß-Diamino Acids via Direct Asymmetric Mannich Reactions.

α,ß-Diamino acids are important structural motifs and building blocks for numerous bioactive natural products, peptidomimetics, and pharmaceuticals, yet efficient asymmetric synthesis to access these stereoarrays remains a challenge. Herein, we report the development of a pyridoxal 5'-phosphate (PLP)-dependent enzyme that is engineered to catalyze stereoselective Mannich-type reactions between free α-amino acids and enolizable cyclic imines. This biocatalyst enabled one-step asymmetric enzymatic synthesis of the unusual pyrrolidine-containing amino acid L-tambroline at gram-scale with high enantio- and diastereocontrol. Furthermore, this enzymatic platform is capable of utilizing a diverse range of α-amino acids as the Mannich donor and various cyclic imines as the acceptor. By coupling with different imine-generating enzymes, we established versatile biocatalytic cascades and demonstrated a general, concise, versatile, and atom-economic approach to access unprotected α,ß-diamino acids, including structurally complex α,α-disubstituted α,ß-diamino acids with contiguous stereocenters.

Negative supercoils regulate meiotic crossover patterns in budding yeast.

Interference exists ubiquitously in many biological processes. Crossover interference patterns meiotic crossovers, which are required for faithful chromosome segregation and evolutionary adaption. However, what the interference signal is and how it is generated and regulated is unknown. We show that yeast top2 alleles which cannot bind or cleave DNA accumulate a higher level of negative supercoils and show weaker interference. However, top2 alleles which cannot religate the cleaved DNA or release the religated DNA accumulate less negative supercoils and show stronger interference. Moreover, the level of negative supercoils is negatively correlated with crossover interference strength. Furthermore, negative supercoils preferentially enrich at crossover-associated Zip3 regions before the formation of meiotic DNA double-strand breaks, and regions with more negative supercoils tend to have more Zip3. Additionally, the strength of crossover interference and homeostasis change coordinately in mutants. These findings suggest that the accumulation and relief of negative supercoils pattern meiotic crossovers.

N-terminal acetylation promotes synaptonemal complex assembly in C. elegans.

N-terminal acetylation of the first two amino acids on proteins is a prevalent cotranslational modification. Despite its abundance, the biological processes associated with this modification are not well understood. Here, we mapped the pattern of protein N-terminal acetylation in Caenorhabditis elegans, uncovering a conserved set of rules for this protein modification and identifying substrates for the N-terminal acetyltransferase B (NatB) complex. We observed an enrichment for global protein N-terminal acetylation and also specifically for NatB substrates in the nucleus, supporting the importance of this modification for regulating biological functions within this cellular compartment. Peptide profiling analysis provides evidence of cross-talk between N-terminal acetylation and internal modifications in a NAT substrate-specific manner. In vivo studies indicate that N-terminal acetylation is critical for meiosis, as it regulates the assembly of the synaptonemal complex (SC), a proteinaceous structure ubiquitously present during meiosis from yeast to humans. Specifically, N-terminal acetylation of NatB substrate SYP-1, an SC structural component, is critical for SC assembly. These findings provide novel insights into the biological functions of N-terminal acetylation and its essential role during meiosis.

Cofactor-independent C-C bond cleavage reactions catalyzed by the AlpJ family of oxygenases in atypical angucycline biosynthesis.

Biosynthesis of atypical angucyclines involves unique oxidative B-ring cleavage and rearrangement reactions, which are catalyzed by AlpJ-family oxygenases, including AlpJ, JadG, and GilOII. Prior investigations established the essential requirement for FADH2/FMNH2 as cofactors when utilizing the quinone intermediate dehydrorabelomycin as a substrate. In this study, we unveil a previously unrecognized facet of these enzymes as cofactor-independent oxygenases when employing the hydroquinone intermediate CR1 as a substrate. The enzymes autonomously drive oxidative ring cleavage and rearrangement reactions of CR1, yielding products identical to those observed in cofactor-dependent reactions of AlpJ-family oxygenases. Furthermore, the AlpJ- and JadG-catalyzed reactions of CR1 could be quenched by superoxide dismutase, supporting a catalytic mechanism wherein the substrate CR1 reductively activates molecular oxygen, generating a substrate radical and the superoxide anion O2 •-. Our findings illuminate a substrate-controlled catalytic mechanism of AlpJ-family oxygenases, expanding the realm of cofactor-independent oxygenases. Notably, AlpJ-family oxygenases stand as a pioneering example of enzymes capable of catalyzing oxidative reactions in either an FADH2/FMNH2-dependent or cofactor-independent manner.

Stereoselective Biocatalytic α-Deuteration of L-Amino Acids by a Pyridoxal 5'-Phosphate-Dependent Mannich Cyclase.

α-Deuterated amino acids are valuable building blocks for developing deuterated drugs, and are important tools for studying biological systems. Biocatalytic deuteration represents an attractive strategy to directly access enantiopure α-deuterated amino acids. Here, we show that a PLP-dependent Mannich cyclase, LolT, involved in the biosynthesis of loline alkaloids, is capable of deuterating a diverse range of L-amino acids, including basic and acidic, nonpolar and polar, aliphatic and aromatic amino acids. Furthermore, complete deuteration of many amino acids can be achieved within minutes with exquisite control on the site- and stereoselectivity. During the course of this investigation, we also unexpectedly discovered that LolT exhibits ß-elimination activity with L-cystine and O-acetyl-L-serine, confirming our previous hypothesis based on structural and phylogenetic analysis that LolT, a Cα-C bond forming enzyme, is evolved from a primordial Cß-S lyase family. Overall, our study demonstrates that LolT is an extremely versatile biocatalyst, and can be used for not only heterocyclic quaternary amino acid biosynthesis, but also biocatalytic amino acid deuteration.

The key players of parthanatos: opportunities for targeting multiple levels in the therapy of parthanatos-based pathogenesis.

Parthanatos is a form of regulated cell death involved in the pathogenesis of many diseases, particularly neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Parthanatos is a multistep cell death pathway cascade that involves poly (ADP-ribose) polymerase 1 (PARP-1) overactivation, PAR accumulation, PAR binding to apoptosis-inducing factor (AIF), AIF release from the mitochondria, nuclear translocation of the AIF/macrophage migration inhibitory factor (MIF) complex, and MIF-mediated large-scale DNA fragmentation. All the key players in the parthanatos pathway are pleiotropic proteins with diverse functions. An in-depth understanding of the structure-based activity of the key factors, and the biochemical mechanisms of parthanatos, is crucial for the development of drugs and therapeutic strategies. In this review, we delve into the key players of the parthanatos pathway and reveal the multiple levels of therapeutic opportunities for treating parthanatos-based pathogenesis.

The demethylase NMAD-1 regulates DNA replication and repair in the Caenorhabditis elegans germline.

The biological roles of nucleic acid methylation, other than at the C5-position of cytosines in CpG dinucleotides, are still not well understood. Here, we report genetic evidence for a critical role for the putative DNA demethylase NMAD-1 in regulating meiosis in C. elegans. nmad-1 mutants have reduced fertility. They show defects in prophase I of meiosis, which leads to reduced embryo production and an increased incidence of males due to defective chromosomal segregation. In nmad-1 mutant worms, nuclear staging beginning at the leptotene and zygotene stages is disorganized, the cohesin complex is mislocalized at the diplotene and diakinesis stages, and chromosomes are improperly condensed, fused, or lost by the end of diakinesis. RNA sequencing of the nmad-1 germline revealed reduced induction of DNA replication and DNA damage response genes during meiosis, which was coupled with delayed DNA replication, impaired DNA repair and increased apoptosis of maturing oocytes. To begin to understand how NMAD-1 regulates DNA replication and repair, we used immunoprecipitation and mass spectrometry to identify NMAD-1 binding proteins. NMAD-1 binds to multiple proteins that regulate DNA repair and replication, including topoisomerase TOP-2 and co-localizes with TOP-2 on chromatin. Moreover, the majority of TOP-2 binding to chromatin depends on NMAD-1. These results suggest that NMAD-1 functions at DNA replication sites to regulate DNA replication and repair during meiosis.

Zipping and Unzipping: Protein Modifications Regulating Synaptonemal Complex Dynamics.

The proteinaceous zipper-like structure known as the synaptonemal complex (SC), which forms between pairs of homologous chromosomes during meiosis from yeast to humans, plays important roles in promoting interhomolog crossover formation, regulating cessation of DNA double-strand break (DSB) formation following crossover designation, and ensuring accurate meiotic chromosome segregation. Recent studies are starting to reveal critical roles for different protein modifications in regulating SC dynamics. Protein SUMOylation, N-terminal acetylation, and phosphorylation have been shown to be essential for the regulated assembly and disassembly of the SC. Moreover, phosphorylation of specific SC components has been found to link changes in SC dynamics with meiotic recombination. This review highlights the latest findings on how protein modifications regulate SC dynamics and functions.

Control of Chromatin Organization and Chromosome Behavior during the Cell Cycle through Phase Separation.

Phase-separated condensates participate in various biological activities. Liquid-liquid phase separation (LLPS) can be driven by collective interactions between multivalent and intrinsically disordered proteins. The manner in which chromatin-with various morphologies and activities-is organized in a complex and small nucleus still remains to be fully determined. Recent findings support the claim that phase separation is involved in the regulation of chromatin organization and chromosome behavior. Moreover, phase separation also influences key events during mitosis and meiosis. This review elaborately dissects how phase separation regulates chromatin and chromosome organization and controls mitotic and meiotic chromosome behavior.

CYLD deficiency promotes pancreatic cancer development by causing mitotic defects.

Successful treatment of pancreatic cancer, which has the highest mortality rate among all types of malignancies, has challenged oncologists for decades, and early detection would undoubtedly increase favorable patient outcomes. The identification of proteins involved in pancreatic cancer progression could lead to biomarkers for early detection of this disease. This study identifies one potential candidate, cylindromatosis (CYLD), a deubiquitinase and microtubule-binding protein that plays a suppressive role in pancreatic cancer development. In pancreatic cancer samples, downregulation of CYLD expression resulted from a loss in the copy number of the CYLD gene; additionally, reduced expression of CYLD negatively correlated with the clinicopathological parameters. Further study demonstrated that CYLD deficiency promoted colony formation in vitro and pancreatic cancer growth in vivo. Mechanistic studies revealed that CYLD is essential for spindle orientation and properly oriented cell division; CYLD deficiency resulted in a substantial increase in chromosome missegregation. Taken together, these data indicate a critical role for CYLD in suppressing pancreatic tumorigenesis, implicating its potential as a biomarker for early detection of pancreatic cancer and a prognostic indicator of patient outcomes.

NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

CYLD regulates spindle orientation by stabilizing astral microtubules and promoting dishevelled-NuMA-dynein/dynactin complex formation.

Oriented cell division is critical for cell fate specification, tissue organization, and tissue homeostasis, and relies on proper orientation of the mitotic spindle. The molecular mechanisms underlying the regulation of spindle orientation remain largely unknown. Herein, we identify a critical role for cylindromatosis (CYLD), a deubiquitinase and regulator of microtubule dynamics, in the control of spindle orientation. CYLD is highly expressed in mitosis and promotes spindle orientation by stabilizing astral microtubules and deubiquitinating the cortical polarity protein dishevelled. The deubiquitination of dishevelled enhances its interaction with nuclear mitotic apparatus, stimulating the cortical localization of nuclear mitotic apparatus and the dynein/dynactin motor complex, a requirement for generating pulling forces on astral microtubules. These findings uncover CYLD as an important player in the orientation of the mitotic spindle and cell division and have important implications in health and disease.

The dynamic recruitment of LAB proteins senses meiotic chromosome axis differentiation in C. elegans.

During meiosis, cohesin and meiosis-specific proteins organize chromatin into an axis-loop architecture, coordinating homologous synapsis, recombination, and ordered chromosome segregation. However, how the meiotic chromosome axis is assembled and differentiated with meiotic progression remains elusive. Here, we explore the dynamic recruitment of two long arms of the bivalent proteins, LAB-1 and LAB-2, in Caenorhabditis elegans. LAB proteins directly interact with the axis core HORMA complexes and weak interactions contribute to their recruitment. LAB proteins phase separate in vitro, and this capacity is promoted by HORMA complexes. During early prophase, synapsis oppositely regulates the axis enrichment of LAB proteins. After the pachytene exit, LAB proteins switch from a reciprocal localization pattern to a colocalization pattern, and the normal dynamic pattern of LAB proteins is altered in meiotic mutants. We propose that LAB recruitment senses axis differentiation, and phase separation of meiotic structures helps subdomain establishment and accurate segregation of the chromosomes.

Reproductive aging: biological pathways and potential interventive strategies.

Reproductive aging is a natural process conserved across species and is well-known in females. It shows age-related follicle depletion and reduction of oocyte quality, eventually causing reproductive senescence and menopause. Although reproductive aging in males is not well noticed as in females, it also causes infertility and has deleterious consequences on the offspring. Various factors have been suggested to contribute to reproductive aging, including oxidative stress, mitochondrial defects, telomere shortening, meiotic chromosome segregation errors and genetic alterations. With the increasing trend of pregnancy age, it is particularly crucial to find interventions to preserve or extend human fertility. Studies in humans and model organisms have provided insights into the biological pathways associated with reproductive aging, and a series of potential interventive strategies have been tested. Here, we review factors affecting reproductive aging in females and males and summarize interventive strategies that may help delay or rescue the aging phenotypes of reproduction.

Phase separation in controlling meiotic chromosome dynamics.

Sexually reproducing organisms produce haploid gametes through meiotic cell division, during which a single round of DNA replication is followed by two consecutive chromosome segregation. A series of meiosis-specific events take place during the meiotic prophase to ensure successful chromosome segregation. These events include programmed DNA double-strand break formation, chromosome movement driven by cytoplasmic forces, homologous pairing, synaptonemal complex installation, and inter-homolog crossover formation. Phase separation has emerged as a key principle controlling cellular biomolecular material organization and biological processes. Recent studies have revealed the involvements of phase separation in assembling meiotic chromosome-associated structures. Here we review and discuss how phase separation may participate in meiotic chromosome dynamics and propose that it may provide opportunities to understand the mysteries in meiotic regulations.

ATF7IP2, a meiosis-specific partner of SETDB1, is required for proper chromosome remodeling and crossover formation during spermatogenesis.

Meiotic crossovers are required for the faithful segregation of homologous chromosomes and to promote genetic diversity. However, it is unclear how crossover formation is regulated, especially on the XY chromosomes, which show a homolog only at the tiny pseudoautosomal region. Here, we show that ATF7IP2 is a meiosis-specific ortholog of ATF7IP and a partner of SETDB1. In the absence of ATF7IP2, autosomes show increased axis length and more crossovers; however, many XY chromosomes lose the obligatory crossover, although the overall XY axis length is also increased. Additionally, meiotic DNA double-strand break formation/repair may also be affected by altered histone modifications. Ultimately, spermatogenesis is blocked, and male mice are infertile. These findings suggest that ATF7IP2 constraints autosomal axis length and crossovers on autosomes; meanwhile, it also modulates XY chromosomes to establish meiotic sex chromosome inactivation for cell-cycle progression and to ensure XY crossover formation during spermatogenesis.

A novel microtubule-modulating agent EM011 inhibits angiogenesis by repressing the HIF-1α axis and disrupting cell polarity and migration.

Endothelial tubular morphogenesis relies on an exquisite interplay of microtubule dynamics and actin remodeling to propel directed cell migration. Recently, the dynamicity and integrity of microtubules have been implicated in the trafficking and efficient translation of the mRNA for HIF-1α (hypoxia-inducible factor), the master regulator of tumor angiogenesis. Thus, microtubule-disrupting agents that perturb the HIF-1α axis and neovascularization cascade are attractive anticancer drug candidates. Here we show that EM011 (9-bromonoscapine), a microtubule-modulating agent, inhibits a spectrum of angiogenic events by interfering with endothelial cell invasion, migration and proliferation. Employing green-fluorescent transgenic zebrafish, we found that EM011 not only inhibited vasculogenesis but also disrupted preexisting vasculature. Mechanistically, EM011 caused proteasome-dependent, VHL-independent HIF-1α degradation and repressed expression of HIF-1α downstream targets, namely VEGF and survivin. Furthermore, EM011 inhibited membrane ruffling and impeded formation of filopodia, lamellipodia and stress fibers, which are critical for cell migration. These events were associated with a drug-mediated decrease in activation of Rho GTPases- RhoA, Cdc42 and Rac1, and correlated with a loss in the geometric precision of centrosome reorientation in the direction of movement. This is the first report to describe a previously unrecognized, antiangiogenic property of a noscapinoid, EM011, and provides evidence for novel anticancer strategies recruited by microtubule-modulating drugs.

CYLD regulates angiogenesis by mediating vascular endothelial cell migration.

Cylindromatosis (CYLD) is a deubiquitinase that was initially identified as a tumor suppressor and has recently been implicated in diverse normal physiologic processes. In this study, we have investigated the involvement of CYLD in angiogenesis, the formation of new blood vessels from preexisting ones. We find that knockdown of CYLD expression significantly impairs angiogenesis in vitro in both matrigel-based tube formation assay and collagen-based 3-dimensional capillary sprouting assay. Disruption of CYLD also remarkably inhibits angiogenic response in vivo, as evidenced by diminished blood vessel growth into the angioreactors implanted in mice. Mechanistic studies show that CYLD regulates angiogenesis by mediating the spreading and migration of vascular endothelial cells. Silencing of CYLD dramatically decreases microtubule dynamics in endothelial cells and inhibits endothelial cell migration by blocking the polarization process. Furthermore, we identify Rac1 activation as an important factor contributing to the action of CYLD in regulating endothelial cell migration and angiogenesis. Our findings thus uncover a previously unrecognized role for CYLD in the angiogenic process and provide a novel mechanism for Rac1 activation during endothelial cell migration and angiogenesis.