Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD interaction domain and a CTD-mimicking region in Sen1

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1

WRN Exonuclease activity is blocked by specific oxidatively induced base lesions positioned in either DNA strand

Werner syndrome (WS) is a premature aging disorder caused by mutations in the WS gene (WRN). Although WRN has been suggested to play an important role in DNA metabolic pathways, such as recombination, replication and repair, its precise role still remains to be determined. WRN possesses ATPase, helicase and exonuclease activities. Previous studies have shown that the WRN exonuclease is inhibited in vitro by certain lesions induced by oxidative stress and positioned in the digested strand of the substrate. The presence of the 70/86 Ku heterodimer (Ku), participating in the repair of double-strand breaks (DSBs), alleviates WRN exonuclease blockage imposed by the oxidatively induced DNA lesions. The current study demonstrates that WRN exonuclease is inhibited by several additional oxidized bases, and that Ku stimulates the WRN exonuclease to bypass these lesions. Specific lesions present in the non-digested strand were shown also to inhibit the progression of the WRN exonuclease; however, Ku was not able to stimulate WRN exonuclease to bypass these lesions. Thus, this study considerably broadens the spectrum of lesions which block WRN exonuclease progression, shows a blocking effect of lesions in the non-digested strand, and supports a function for WRN and Ku in a DNA damage processing pathway

Medium-Chain Polyprenols Influence Chloroplast Membrane Dynamics In Solanum Lycopersicum

The widespread occurrence of polyprenols throughout the plant kingdom is well documented, yet their functional role is poorly understood. These lipophilic compounds are known to be assembled from isoprenoid precursors by a class of enzymes designated as cisprenyltransferases (CPTs), which are encoded by small CPT gene families in plants. In this study, we report that RNAi-mediated knockdown of one member of the tomato CPT family (SlCPT5) reduced polyprenols in leaves by ~70%. Assays with recombinant SlCPT5 produced in E. coli determined that the enzyme synthesizes polyprenols of approximately 50-55 carbons (Pren-10, Pren-11) in length and accommodates a variety of trans-prenyldiphosphate precursors as substrates. Introduction of SlCPT5 into the polyprenol-deficient yeast Δrer2 mutant resulted in the accumulation of Pren-11 in yeast cells, restored proper protein Nglycosylation, and rescued the temperature sensitive growth phenotype that is associated with its polyprenol deficiency. Subcellular fractionation studies together with in vivo localization of SlCPT5 fluorescent protein fusions demonstrated that SlCPT5 resides in the chloroplast stroma and that its enzymatic products accumulate into both thylakoid and envelope membranes. Transmission electron microscopy images of polyprenol-deficient leaves revealed alterations in chloroplast ultrastructure and anisotropy measurements revealed a more disordered state of their envelope membranes. In polyprenol-deficient leaves, CO2 assimilation was hindered and their thylakoid membranes exhibited lower phase transition temperatures and calorimetric enthalpies, which coincided with a decreased photosynthetic electron transport rate. Taken together, these results uncover a role for polyprenols in governing chloroplast membrane dynamics

A cap 0-dependent mRNA capture method to analyze the yeast transcriptome

Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes

Metallothionein crypt-restricted immunopositivity indices (MTCRII) correlate with aberrant crypt foci (ACF) in mouse colon

Metallothionein (MT) crypt-restricted immunopositivity indices (MTCRII) are colonic crypt stem cell mutation markers that may be induced early and in abundance after mutagen treatment. Metallothionein is the endogenous reporter gene for MTCRII, but is not typically implicated in the classical pathway of colorectal tumorigenesis. Hence, the oncological relevance of MTCRII is unclear. This study tests the hypothesis that MTCRII induced by N-methyl-N-nitrosourea (MNU) and lambda carrageenan (λCgN) associate with aberrant crypt foci (ACF) in mouse colon. Undegraded λCgN and MNU were tested alone and in combination against MTCRII and ACF in Balb/c mice, at 20 weeks after the start of treatment. MTCRII were unaffected by λCgN alone. Combined λCgN/MNU treatments induced greater MTCRII (P<0.01) as well as greater number (P<0.001) and crypt multiplicity (P<0.01) of ACF than MNU alone. MTCRII were approximately 10-fold more numerous than ACF, although linear correlations were observed between these parameters (r=0.732; P<0.01). MTCRII are induced by λCgN/MNU interactions in sufficient numbers to provide statistical power from relatively small sample sizes and correlate with ACF formation. MTCRII could thus provide the basis for a novel medium-term murine bioassay relevant to early-stage colorectal tumorigenesis

The yield of essential oils in Melaleuca alternifolia (Myrtaceae) is regulated through transcript abundance of genes in the MEP pathway

Medicinal tea tree (Melaleuca alternifolia) leaves contain large amounts of an essential oil, dominated by monoterpenes. Several enzymes of the chloroplastic methylerythritol phosphate (MEP) pathway are hypothesised to act as bottlenecks to the production of monoterpenes. We investigated, whether transcript abundance of genes encoding for enzymes of the MEP pathway were correlated with foliar terpenes in M. alternifolia using a population of 48 individuals that ranged in their oil concentration from 39 -122 mg x g DM(-1). Our study shows that most genes in the MEP pathway are co-regulated and that the expression of multiple genes within the MEP pathway is correlated with oil yield. Using multiple regression analysis, variation in expression of MEP pathway genes explained 87% of variation in foliar monoterpene concentrations. The data also suggest that sesquiterpenes in M. alternifolia are synthesised, at least in part, from isopentenyl pyrophosphate originating from the plastid via the MEP pathway

In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay

Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P < 0.05). B[a]P-related DNA adducts were detected at increased levels in smokers as determined by immunostaining. Direct exposure of mature sperm cells to B[a]P (10 or 25 μM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 μM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE–DNA adducts as measured with 32P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies

Spontaneous DNA damage to the nuclear genome promotes senescence,redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Spontaneous DNA damage to the nuclear genome promotes senescence, T redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R mutation abolished the interaction with POLβ, but did not disrupt the interactions with PARP-1, LIG3α and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLβ interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S) and two frequent (R194W and R399Q) amino acid population variants had little or no effect on XRCC1 protein stability or the interactions with POLβ, PARP-1, LIG3α, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLβ, PARP-1, LIG3α and PCNA, but did reduce DNA-binding ability. When expressed in HeLa cells, the XRCC1 variants—excluding E98K, which was largely nucleolar, and C389Y, which exhibited reduced expression—exhibited normal nuclear distribution. Most of the protein variants, including the V86R POLβ-interaction mutant, displayed normal relocalization kinetics to/from sites of laser-induced DNA damage: except for E98K and C389Y, and the polymorphic variant R280H, which exhibited a slightly shorter retention time at DNA breaks