Differential Regulation of Germline Apoptosis in Response to Meiotic Checkpoint Activation

In Caenorhabditis elegans, germline apoptosis is promoted by egl-1 and ced-13 in response to meiotic checkpoint activation. We report that the requirement for these two factors depends on which checkpoints are active. We also identify a regulatory region of egl-1 required to inhibit germline apoptosis in response to DNA damage incurred during meiotic recombination.     

The AAA+ ATPase TRIP13 remodels HORMA domains through N‐terminal engagement and unfolding

Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed “closure motifs”. The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain–closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X‐ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31^comet. We show that p31^comet binding to the TRIP13 N‐terminal domain positions the disordered MAD2 N‐terminus for engagement by the TRIP13 “pore loops”, which then unfold MAD2 in the presence of ATP. N‐terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro, and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13‐mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain–closure motif complexes by TRIP13.     

Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein,Hop1

A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis.     

HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity

1. Download : Download high-res image (118KB)
2. Download : Download full-size image

     

A Meiotic Checkpoint Alters Repair Partner Bias to Permit Inter-sister Repair of Persistent DSBs

1. Download high-res image (194KB)
2. Download full-size image

     

A novel DNA repair inhibitor,diallyl disulfide (DADS), impairs DNA resection during DNA double-strand break repair by reducing Sae2 and Exo1 levels

Combining natural products with chemotherapy and/or radiotherapy may increase the efficacy of cancer treatment. It has been hypothesized that natural products may inhibit DNA repair and sensitize cancer cells to DNA damage-based cancer therapy. However, the molecular mechanisms underlying these activities remain unclear. In this study, we found that diallyl disulfide (DADS), an organosulfur compound, increased the sensitivity of yeast cells to DNA damage and has potential for development as an adjuvant drug for DNA damage-based cancer therapy. We induced HO endonuclease to generate a specific DNA double-strand break (DSB) by adding galactose to yeast and used this system to study how DADS affects DNA repair. In this study, we found that DADS inhibited DNA repair in single-strand annealing (SSA) system and sensitized SSA cells to a single DSB. DADS impaired DNA repair by inhibiting the protein levels of the DNA resection-related proteins Sae2 and Exo1. We also found that the recruitment of MRX and the Mec1-Ddc2 complex to a DSB was prevented by DADS. This result suggests that DADS counteracts G2/M DNA damage checkpoint activation in a Mec1 (ATR)- and Tel1 (ATM)-dependent manner. Only by elucidating the molecular mechanisms by which DADS influences DNA repair will we be able to discover new adjuvant drugs to improve chemotherapy and/or radiotherapy.     

Cytologic and Autoradiographic Studies of the Micronucleus at Meiotic Prophase in Tetrahymena pyriformis

Micronuclear changes of variety 1 of Tetrahymena pyriformis during meiotic prophase have been observed by the light microscope. Morphologic changes in the micronucleus are divided into 6 stages. In stage I, chromatin begins to polarize; in stage II, the micronucleus becomes spindle shaped; and in stage III, one end of the micronucleus protrudes to form a “neck.” In stage IV, where the micronucleus elongates to maximal length, the whole micronucleus consists of 2 chromatin threads pairing longitudinally. One thread probably contains one genome. In stage V, the elongated thread becomes shorter and thicker. Finally, in stage VI, separate chromosomes appear and enter into metaphase. To discover the role of the elongation of the micronucleus, called crescent formation, autoradiographic analysis of RNA and DNA synthesis were undertaken using [³H]uridine and [³H]thymidine. Pulse label and chase experiments show that the crescent in stages II and III is actively synthesizing RNA. Though no remarkable DNA synthesis was observed during meiosis, a small amount of DNA synthesis occurred during the 1st and 2nd prezygotic divisions.     

Pseudosynapsis and Decreased Stringency of Meiotic Repair Pathway Choice on the Hemizygous Sex Chromosome of Caenorhabditis elegans Males

During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes.     

Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombeRad1 ortholog promotes cell survival against DNA damage and is required for G₂/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1^-/- ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G₂/M as well as S/M checkpoints. These data indicated that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G₂/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.     

Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of Schizosaccharomyces pombe AAA+ ATPase Abo1

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.     

Ciprofloxacin is an inhibitor of the Mcm2-7 replicative helicase

Most currently available small molecule inhibitors of DNA replication lack enzymatic specificity, resulting in deleterious side effects during use in cancer chemotherapy and limited experimental usefulness as mechanistic tools to study DNA replication. Towards development of targeted replication inhibitors, we have focused on Mcm2-7 (minichromosome maintenance protein 2–7), a highly conserved helicase and key regulatory component of eukaryotic DNA replication. Unexpectedly we found that the fluoroquinolone antibiotic ciprofloxacin preferentially inhibits Mcm2-7. Ciprofloxacin blocks the DNA helicase activity of Mcm2-7 at concentrations that have little effect on other tested helicases and prevents the proliferation of both yeast and human cells at concentrations similar to those that inhibit DNA unwinding. Moreover, a previously characterized mcm mutant (mcm4chaos3) exhibits increased ciprofloxacin resistance. To identify more potent Mcm2-7 inhibitors, we screened molecules that are structurally related to ciprofloxacin and identified several that compromise the Mcm2-7 helicase activity at lower concentrations. Our results indicate that ciprofloxacin targets Mcm2-7 in vitro, and support the feasibility of developing specific quinolone-based inhibitors of Mcm2-7 for therapeutic and experimental applications.     

Crystal structure of the N‐terminal domain of EccA1 ATPase from the ESX‐1 secretion system of Mycobacterium tuberculosis

EccA₁ is an important component of the type VII secretion system (T7SS) that is responsible for transport of virulence factors in pathogenic mycobacteria. EccA₁ has an N‐terminal domain of unknown function and a C‐terminal AAA+ (ATPases associated with various cellular activities) domain. Here we report the crystal structure of the N‐terminal domain of EccA₁ from Mycobacterium tuberculosis, which shows an arrangement of six tetratricopeptide repeats that may mediate interactions of EccA₁ with secreted substrates. Furthermore, the size and shape of the N‐terminal domain suggest its orientation in the context of a hexamer model of full‐length EccA₁. Proteins 2014; 82:159–163. © 2013 Wiley Periodicals, Inc.     

Functional Relationship of ATP Hydrolysis,Presynaptic Filament Stability,and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1

DMC1 and RAD51 are conserved recombinases that catalyze homologous recombination. DMC1 and RAD51 share similar properties in DNA binding, DNA-stimulated ATP hydrolysis, and catalysis of homologous DNA strand exchange. A large body of evidence indicates that attenuation of ATP hydrolysis leads to stabilization of the RAD51-ssDNA presynaptic filament and enhancement of DNA strand exchange. However, the functional relationship of ATPase activity, presynaptic filament stability, and DMC1-mediated homologous DNA strand exchange has remained largely unexplored. To address this important question, we have constructed several mutant variants of human DMC1 and characterized them biochemically to gain mechanistic insights. Two mutations, K132R and D223N, that change key residues in the Walker A and B nucleotide-binding motifs ablate ATP binding and render DMC1 inactive. On the other hand, the nucleotide-binding cap D317K mutant binds ATP normally but shows significantly attenuated ATPase activity and, accordingly, forms a highly stable presynaptic filament. Surprisingly, unlike RAD51, presynaptic filament stabilization achieved via ATP hydrolysis attenuation does not lead to any enhancement of DMC1-catalyzed homologous DNA pairing and strand exchange. This conclusion is further supported by examining wild-type DMC1 with non-hydrolyzable ATP analogues. Thus, our results reveal an important mechanistic difference between RAD51 and DMC1.     

Meiotic recombination mirrors patterns of germline replication in mice and humans

1. Download : Download high-res image (241KB)
2. Download : Download full-size image

     

Wing 1 of protein HOP2 is as important as helix 3 in DNA binding by MD simulation

The repair of programmed DNA double-strand breaks through recombination is required for proper association and disjunction of the meiotic homologous chromosomes. Meiosis-specific protein HOP2 plays essential roles in recombination by promoting recombinase activities. The N-terminal domain of HOP2 interacts with DNA through helix 3 (H3) and wing 1 (W1). Mutations in wing 1 (Y65A/K67A/Q68A) slightly weakened the binding but mutations in helices 2 and 3 (Q30A/K44A/K49A) nearly abolished the binding. To better understand such differential effects at atomic level, molecular dynamics simulations were employed. Despite losing some hydrogen bonds, the W1-mutant DNA complex was rescued by stronger hydrophobic interactions. For the wild type and W1-mutant, the protein was found to slide along the DNA grooves as the DNA rolls along its double-helix axis. This motion could be functionally important to facilitate the precise positioning of the single-stranded DNA with the homologous double-stranded DNA. The sliding motion was reduced in the W1-mutant. The H-mutant nearly lost all intermolecular interactions. Moreover, an additional mutation in wing 1 (Y65A/K67A/Q68A/K69A) also caused complete complex dissociation. Therefore, both wing 1 and helix 3 make important contribution to the DNA binding, which could be important to the strand invasion function of HOP2 homodimer and HOP2-MND1 heterodimer. Similar to cocking a medieval crossbow with the archer’s foot placed in the stirrup, wing 1 may push the minor groove to cause distortion while helix 3 grabs the major groove.     

Msh4 and Msh5 Function in SC-Independent Chiasma Formation During the Streamlined Meiosis of Tetrahymena

ZMM proteins have been defined in budding yeast as factors that are collectively involved in the formation of interfering crossovers (COs) and synaptonemal complexes (SCs), and they are a hallmark of the predominant meiotic recombination pathway of most organisms. In addition to this so-called class I CO pathway, a minority of crossovers are formed by a class II pathway, which involves the Mus81-Mms4 endonuclease complex. This is the only CO pathway in the SC-less meiosis of the fission yeast. ZMM proteins (including SC components) were always found to be co-occurring and hence have been regarded as functionally linked. Like the fission yeast, the protist Tetrahymena thermophila does not possess a SC, and its COs are dependent on Mus81-Mms4. Here we show that the ZMM proteins Msh4 and Msh5 are required for normal chiasma formation, and we propose that they have a pro-CO function outside a canonical class I pathway in Tetrahymena. Thus, the two-pathway model is not tenable as a general rule.     

The Essential Function of SETDB1 in Homologous Chromosome Pairing and Synapsis during Meiosis

1. Download : Download high-res image (192KB)
2. Download : Download full-size image

     

A conserved function for a Caenorhabditis elegans Com1/Sae2/CtIP protein homolog in meiotic recombination

Genome stability relies on faithful DNA repair both in mitosis and in meiosis. Here, we report on a Caenorhabditis elegans protein that we found to be homologous to the mammalian repair-related protein CtIP and to the budding yeast Com1/Sae2 recombination protein. A com-1 mutant displays normal meiotic chromosome pairing but forms irregular chromatin aggregates instead of diakinesis bivalents. While meiotic DNA double-strand breaks (DSBs) are formed, they appear to persist or undergo improper repair. Despite the presence of DSBs, the recombination protein RAD-51, which is known to associate with single-stranded DNA (ssDNA) flanking DSBs, does not localize to meiotic chromosomes in the com-1 mutant. Exposure of the mutant to gamma-radiation, however, induces RAD-51 foci, which suggests that the failure of RAD-51 to load is specific to meiotic (SPO-11-generated) DSBs. These results suggest that C. elegans COM-1 plays a role in the generation of ssDNA tails that can load RAD-51, invade homologous DNA tracts and thereby initiate recombination. Extrapolating from the worm homolog, we expect similar phenotypes for mutations in the mammalian tumor suppressor CtIP.