Yeast ORC sumoylation status fine-tunes origin licensing.

Sumoylation is emerging as a posttranslation modification important for regulating chromosome duplication and stability. The origin recognition complex (ORC) that directs DNA replication initiation by loading the MCM replicative helicase onto origins is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are unclear. Here we report the effects of hypersumoylation and hyposumoylation of yeast ORC on ORC activity and origin function using multiple approaches. ORC hypersumoylation preferentially reduced the function of a subset of early origins, while Orc2 hyposumoylation had an opposing effect. Mechanistically, ORC hypersumoylation reduced MCM loading in vitro and diminished MCM chromatin association in vivo. Either hypersumoylation or hyposumoylation of ORC resulted in genome instability and the dependence of yeast on other genome maintenance factors, providing evidence that appropriate ORC sumoylation levels are important for cell fitness. Thus, yeast ORC sumoylation status must be properly controlled to achieve optimal origin function across the genome and genome stability.

Regulated Proteolysis of MutSγ Controls Meiotic Crossing Over.

Crossover recombination is essential for accurate chromosome segregation during meiosis. The MutSγ complex, Msh4-Msh5, facilitates crossing over by binding and stabilizing nascent recombination intermediates. We show that these activities are governed by regulated proteolysis. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable by directly targeting proteasomal degradation. Activation of MutSγ requires the Dbf4-dependent kinase Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Genetic requirements for Msh4 phosphorylation indicate that DDK targets MutSγ only after it has bound to nascent joint molecules (JMs) in the context of synapsing chromosomes. Overexpression studies confirm that the steady-state level of Msh4, not phosphorylation per se, is the critical determinant for crossing over. At the DNA level, Msh4 phosphorylation enables the formation and crossover-biased resolution of double-Holliday Junction intermediates. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossing over.

Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase.

At the final step of homologous recombination, Holliday junction-containing joint molecules (JMs) are resolved to form crossover or noncrossover products. The enzymes responsible for JM resolution in vivo remain uncertain, but three distinct endonucleases capable of resolving JMs in vitro have been identified: Mus81-Mms4(EME1), Slx1-Slx4(BTBD12), and Yen1(GEN1). Using physical monitoring of recombination during budding yeast meiosis, we show that all three endonucleases are capable of promoting JM resolution in vivo. However, in mms4 slx4 yen1 triple mutants, JM resolution and crossing over occur efficiently. Paradoxically, crossing over in this background is strongly dependent on the Blooms helicase ortholog Sgs1, a component of a well-characterized anticrossover activity. Sgs1-dependent crossing over, but not JM resolution per se, also requires XPG family nuclease Exo1 and the MutLγ complex Mlh1-Mlh3. Thus, Sgs1, Exo1, and MutLγ together define a previously undescribed meiotic JM resolution pathway that produces the majority of crossovers in budding yeast and, by inference, in mammals.

PCNA activates the MutLγ endonuclease to promote meiotic crossing over.

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation1. Defective crossing over causes infertility, miscarriage and congenital disease. Each pair of chromosomes attains at least one crossover via the formation and biased resolution of recombination intermediates known as double Holliday junctions2,3. A central principle of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands4. The endonuclease activity of the MutLγ complex has been implicated in the resolution of crossovers5-10, but the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp PCNA is important for crossover-biased resolution. In vitro assays with human enzymes show that PCNA and its loader RFC are sufficient to activate the MutLγ endonuclease. MutLγ is further stimulated by a co-dependent activity of the pro-crossover factors EXO1 and MutSγ, the latter of which binds Holliday junctions11. MutLγ also binds various branched DNAs, including Holliday junctions, but does not show canonical resolvase activity, implying that the endonuclease incises adjacent to junction branch points to achieve resolution. In vivo, RFC facilitates MutLγ-dependent crossing over in budding yeast. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover resolution and the initiation steps of DNA mismatch repair12,13 and evoke a novel model for crossover-specific resolution of double Holliday junctions during meiosis.

Impeding DNA Break Repair Enables Oocyte Quality Control.

Oocyte quality control culls eggs with defects in meiosis. In mouse, oocyte death can be triggered by defects in chromosome synapsis and recombination, which involve repair of DNA double-strand breaks (DSBs) between homologous chromosomes. We show that RNF212, a SUMO ligase required for crossing over, also mediates oocyte quality control. Both physiological apoptosis and wholesale oocyte elimination in meiotic mutants require RNF212. RNF212 sensitizes oocytes to DSB-induced apoptosis within a narrow window as chromosomes desynapse and cells transition into quiescence. Analysis of DNA damage during this transition implies that RNF212 impedes DSB repair. Consistently, RNF212 is required for HORMAD1, a negative regulator of inter-sister recombination, to associate with desynapsing chromosomes. We infer that oocytes impede repair of residual DSBs to retain a "memory" of meiotic defects that enables quality-control processes. These results define the logic of oocyte quality control and suggest RNF212 variants may influence transmission of defective genomes.

Cellular self-organization: An overdrive in Cambrian diversity?

During the early Cambrian period metazoan life forms diverged at an accelerated rate to occupy multiple ecological niches on earth. A variety of explanations have been proposed to address this major evolutionary phenomenon termed the "Cambrian explosion." While most hypotheses address environmental, developmental, and ecological factors that facilitated evolutionary innovations, the biological basis for accelerated emergence of species diversity in the Cambrian period remains largely conjectural. Herein, we posit that morphogenesis by self-organization enables the uncoupling of genomic mutational landscape from phenotypic diversification. Evidence is provided for a two-tiered interpretation of genomic changes in metazoan animals wherein mutations not only impact upon function of individual cells, but also alter the self-organization outcome during developmental morphogenesis. We provide evidence that the morphological impacts of mutations on self-organization could remain repressed if associated with an unmet negative energetic cost. We posit that accelerated morphological diversification in transition to the Cambrian period has occurred by emergence of dormant (i.e., reserved) morphological novelties whose molecular underpinnings were seeded in the Precambrian period.

Hop1 and the meiotic DNA-damage response.

During the DNA-damage response, adaptor proteins mediate signaling between the PI3K-like sensor kinases, ATM and ATR, and serine/threonine effector kinases. Carballo et al. (2008) now show that the chromosomal protein Hop1 mediates PI3K-like kinase signaling during the repair of DNA double-strand breaks (DSBs) in meiosis.

Pervasive and essential roles of the Top3-Rmi1 decatenase orchestrate recombination and facilitate chromosome segregation in meiosis.

The Bloom's helicase ortholog, Sgs1, plays central roles to coordinate the formation and resolution of joint molecule intermediates (JMs) during meiotic recombination in budding yeast. Sgs1 can associate with type-I topoisomerase Top3 and its accessory factor Rmi1 to form a conserved complex best known for its unique ability to decatenate double-Holliday junctions. Contrary to expectations, we show that the strand-passage activity of Top3-Rmi1 is required for all known functions of Sgs1 in meiotic recombination, including channeling JMs into physiological crossover and noncrossover pathways, and suppression of non-allelic recombination. We infer that Sgs1 always functions in the context of the Sgs1-Top3-Rmi1 complex to regulate meiotic recombination. In addition, we reveal a distinct late role for Top3-Rmi1 in resolving recombination-dependent chromosome entanglements to allow segregation at anaphase. Surprisingly, Sgs1 does not share this essential role of Top3-Rmi1. These data reveal an essential and pervasive role for the Top3-Rmi1 decatenase during meiosis.

The fate of notch-1 transcript is linked to cell cycle dynamics by activity of a natural antisense transcript.

A core imprint of metazoan life is that perturbations of cell cycle are offset by compensatory changes in successive cellular generations. This trait enhances robustness of multicellular growth and requires transmission of signaling cues within a cell lineage. Notably, the identity and mode of activity of transgenerational signals remain largely unknown. Here we report the discovery of a natural antisense transcript encoded in exon 25 of notch-1 locus (nAS25) by which mother cells control the fate of notch-1 transcript in daughter cells to buffer against perturbations of cell cycle. The antisense transcript is transcribed at G1 phase of cell cycle from a bi-directional E2F1-dependent promoter in the mother cell where the titer of nAS25 is calibrated to the length of G1. Transmission of the antisense transcript from mother to daughter cells stabilizes notch-1 sense transcript in G0 phase of daughter cells by masking it from RNA editing and resultant nonsense-mediated degradation. In consequence, nAS25-mediated amplification of notch-1 signaling reprograms G1 phase in daughter cells to compensate for the altered dynamics of the mother cell. The function of nAS25/notch-1 in integrating G1 phase history of the mother cell into that of daughter cells is compatible with the predicted activity of a molecular oscillator, slower than cyclins, that coordinates cell cycle within cell lineage.

Notch ankyrin domain: evolutionary rise of a thermodynamic sensor.

Notch signalling pathway plays a key role in metazoan biology by contributing to resolution of binary decisions in the life cycle of cells during development. Outcomes such as proliferation/differentiation dichotomy are resolved by transcriptional remodelling that follows a switch from Notchon to Notchoff state, characterised by dissociation of Notch intracellular domain (NICD) from DNA-bound RBPJ. Here we provide evidence that transitioning to the Notchoff state is regulated by heat flux, a phenomenon that aligns resolution of fate dichotomies to mitochondrial activity. A combination of phylogenetic analysis and computational biochemistry was utilised to disclose structural adaptations of Notch1 ankyrin domain that enabled function as a sensor of heat flux. We then employed DNA-based micro-thermography to measure heat flux during brain development, followed by analysis in vitro of the temperature-dependent behaviour of Notch1 in mouse neural progenitor cells. The structural capacity of NICD to operate as a thermodynamic sensor in metazoans stems from characteristic enrichment of charged acidic amino acids in ß-hairpins of the ankyrin domain that amplify destabilising inter-residue electrostatic interactions and render the domain thermolabile. The instability emerges upon mitochondrial activity which raises the perinuclear and nuclear temperatures to 50 °C and 39 °C, respectively, leading to destabilization of Notch1 transcriptional complex and transitioning to the Notchoff state. Notch1 functions a metazoan thermodynamic sensor that is switched on by intercellular contacts, inputs heat flux as a proxy for mitochondrial activity in the Notchon state via the ankyrin domain and is eventually switched off in a temperature-dependent manner. Video abstract.

Intra-species variation within Lactobacillus rhamnosus correlates to beneficial or harmful outcomes: lessons from the oral cavity.

BACKGROUND: The origin of most of the Lactobacillus rhamnosus genome sequences lodged in NCBI can be traced to food and faecal isolates followed by blood and tissue sites but with minimal representation from oral and vaginal isolates. However, on the L. rhamnosus phylogenetic tree no apparent clade is linked to the origin of isolation or to the relevant clinical source, except for a distinct clade exclusively shared by L. rhamnosus isolates from early stages of dental pulp infection (LRHMDP2 and LRHMDP3) and from bronchoalveolar lavage (699_LRHA and 708_LRHA) from a critical care patient. These L. rhamnosus strains, LRHMDP2, LRHMDP3, 699_LRHA and 708_LRHA isolated from different continents, display closest genome neighbour gapped identity of 99.95%. The aim of this study was to define a potentially unique complement of genes of clinical relevance shared between these L. rhamnosus clinical isolates in comparison to probiotic L. rhamnosus strains. RESULTS: In this analysis we used orthologous protein identification tools such as ProteinOrtho followed by tblastn alignments to identify a novel tyrosine protein phosphatase (wzb)-tyrosine-protein kinase modulator EpsC (wzd)- synteny exopolysaccharide (EPS) cluster. This EPS cluster was specifically conserved in a clade of 5 clinical isolates containing the four L. rhamnosus clinical isolates noted above and Lactobacillus spp. HMSC077C11, a clinical isolate from a neck abscess. The EPS cluster was shared with only two other strains, L. rhamnosus BPL5 and BPL15, which formed a distant clade on the L. rhamnosus phylogenetic tree, with a closest genome neighbour gapped identity of 97.51% with L. rhamnosus LRHMDP2 and LRHMDP3. Exclusivity of this EPS cluster (from those identified before) was defined by five EPS genes, which were specifically conserved between the clade of 5 clinical isolates and L. rhamnosus BPL5 and BPL15 when compared to the remaining L. rhamnosus strains. Comparative genome analysis between the clade of 5 clinical isolates and L. rhamnosus BPL5 and BPL15 showed a set of 58 potentially unique genes characteristic of the clade of 5. CONCLUSION: The potentially unique functional protein orthologs associated with the clade of 5 clinical isolates may provide understanding of fitness under selective pressure.

Maternal obesity enhances oocyte chromosome abnormalities associated with aging.

Obesity is increasing globally, and maternal obesity has adverse effects on pregnancy outcomes and the long-term health of offspring. Maternal obesity has been associated with pregnancy failure through impaired oogenesis and embryogenesis. However, whether maternal obesity causes chromosome abnormalities in oocytes has remained unclear. Here we show that chromosome abnormalities are increased in the oocytes of obese mice fed a high-fat diet and identify weakened sister-chromatid cohesion as the likely cause. Numbers of full-grown follicles retrieved from obese mice were the same as controls and the efficiency of in vitro oocyte maturation remained high. However, chromosome abnormalities presenting in both metaphase-I and metaphase-II were elevated, most prominently the premature separation of sister chromatids. Weakened sister-chromatid cohesion in oocytes from obese mice was manifested both as the terminalization of chiasmata in metaphase-I and as increased separation of sister centromeres in metaphase II. Obesity-associated abnormalities were elevated in older mice implying that maternal obesity exacerbates the deterioration of cohesion seen with advancing age.

Notch pathway: a bistable inducer of biological noise?

Notch signalling pathway is central to development of metazoans. The pathway codes a binary fate switch. Upon activation, downstream signals contribute to resolution of fate dichotomies such as proliferation/differentiation or sub-lineage differentiation outcome. There is, however, an interesting paradox in the Notch signalling pathway. Despite remarkable predictability of fate outcomes instructed by the Notch pathway, the associated transcriptome is versatile and plastic. This inconsistency suggests the presence of an interface that compiles input from the plastic transcriptome of the Notch pathway but communicates only a binary output in biological decisions. Herein, we address the interface that determines fate outcomes. We provide an alternative hypothesis for the Notch pathway as a biological master switch that operates by induction of genetic noise and bistability in order to facilitate resolution of dichotomous fate outcomes in development.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

Metronidazole: an update on metabolism, structure-cytotoxicity and resistance mechanisms.

Metronidazole, a nitroimidazole, remains a front-line choice for treatment of infections related to inflammatory disorders of the gastrointestinal tract including colitis linked to Clostridium difficile. Despite >60 years of research, the metabolism of metronidazole and associated cytotoxicity is not definitively characterized. Nitroimidazoles are prodrugs that are reductively activated (the nitro group is reduced) under low oxygen tension, leading to imidazole fragmentation and cytotoxicity. It remains unclear if nitroimidazole reduction (activation) contributes to the cytotoxicity profile, or whether subsequent fragmentation of the imidazole ring and formed metabolites alone mediate cytotoxicity. A molecular mechanism underpinning high level (>256 mg/L) bacterial resistance to metronidazole also remains elusive. Considering the widespread use of metronidazole and other nitroimidazoles, this review was undertaken to emphasize the structure-cytotoxicity profile of the numerous metabolites of metronidazole in human and murine models and to examine conflicting reports regarding metabolite-DNA interactions. An alternative hypothesis, that DNA synthesis and repair of existing DNA is indirectly inhibited by metronidazole is proposed. Prokaryotic metabolism of metronidazole is detailed to discuss new resistance mechanisms. Additionally, the review contextualizes the history and current use of metronidazole, rates of metronidazole resistance including metronidazole MDR as well as the biosynthesis of azomycin, the natural precursor of metronidazole. Changes in the gastrointestinal microbiome and the host after metronidazole administration are also reviewed. Finally, novel nitroimidazoles and new antibiotic strategies are discussed.

Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions.

The Rad2/XPG family nuclease, Exo1, functions in a variety of DNA repair pathways. During meiosis, Exo1 promotes crossover recombination and thereby facilitates chromosome segregation at the first division. Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs). Nucleolytic resection of DSBs generates long 3' single-strand tails that undergo strand exchange with a homologous chromosome to form joint molecule (JM) intermediates. We show that meiotic DSB resection is dramatically reduced in exo1Δ mutants and test the idea that Exo1-catalyzed resection promotes crossing over by facilitating formation of crossover-specific JMs called double Holliday junctions (dHJs). Contrary to this idea, dHJs form at wild-type levels in exo1Δ mutants, implying that Exo1 has a second function that promotes resolution of dHJs into crossovers. Surprisingly, the dHJ resolution function of Exo1 is independent of its nuclease activities but requires interaction with the putative endonuclease complex, Mlh1-Mlh3. Thus, the DSB resection and procrossover functions of Exo1 during meiosis involve temporally and biochemically distinct activities.

Double Holliday junctions are intermediates of DNA break repair.

Repair of DNA double-strand breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumour suppression. However, despite its importance, the molecular intermediates of mitotic DSB repair remain undefined. The double Holliday junction (DHJ), presupposed to be the central intermediate for more than 25 years, has only been identified during meiotic recombination. Moreover, evidence has accumulated for alternative, DHJ-independent mechanisms, raising the possibility that DHJs are not formed during DSB repair in mitotically cycling cells. Here we identify intermediates of DSB repair by using a budding-yeast assay system designed to mimic physiological DSB repair. This system uses diploid cells and provides the possibility for allelic recombination either between sister chromatids or between homologues, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homologue joint molecule (JM) intermediates whose strand composition and size are identical to those of the canonical DHJ structures observed in meiosis. However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologues. Moreover, JMs seem to represent a minor pathway of DSB repair in mitotic cells, being detected at about tenfold lower levels (per DSB) than during meiotic recombination. Thus, although DHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.

Rad52 promotes postinvasion steps of meiotic double-strand-break repair.

During DNA double-strand-break (DSB) repair by recombination, the broken chromosome uses a homologous chromosome as a repair template. Early steps of recombination are well characterized: DSB ends assemble filaments of RecA-family proteins that catalyze homologous pairing and strand-invasion reactions. By contrast, the postinvasion steps of recombination are poorly characterized. Rad52 plays an essential role during early steps of recombination by mediating assembly of a RecA homolog, Rad51, into nucleoprotein filaments. The meiosis-specific RecA-homolog Dmc1 does not show this dependence, however. By exploiting the Rad52 independence of Dmc1, we reveal that Rad52 promotes postinvasion steps of both crossover and noncrossover pathways of meiotic recombination in Saccharomyces cerevisiae. This activity resides in the N-terminal region of Rad52, which can anneal complementary DNA strands, and is independent of its Rad51-assembly function. Our findings show that Rad52 functions in temporally and biochemically distinct reactions and suggest a general annealing mechanism for reuniting DSB ends during recombination.

RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination.

Saccharomyces cerevisiae RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, are implicated in processing joint molecule (JM) recombination intermediates. We show that cells lacking either enzyme frequently experience chromosome segregation problems during meiosis and that when both enzymes are absent attempted segregation fails catastrophically. In all cases, segregation appears to be impeded by unresolved JMs. Analysis of the DNA events of recombination indicates that Sgs1 limits aberrant JM structures that result from secondary strand-invasion events and often require Mus81-Mms4 for their normal resolution. Aberrant JMs contain high levels of single Holliday junctions and include intersister JMs, multichromatid JMs comprising three and four chromatids, and newly identified recombinant JMs containing two chromatids, one of which has undergone crossing over. Despite persistent JMs in sgs1 mms4 double mutants, crossover and noncrossover products still form at high levels. We conclude that Sgs1 and Mus81-Mms4 collaborate to eliminate aberrant JMs, whereas as-yet-unidentified enzymes process normal JMs.