Nucleic Acids Research

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Mutational analysis of the Potyviridae transcriptional slippage site utilized for expression of the P3N-PIPO and P1N-PISPO proteins

Mon, 2016-09-19 08:02

The Potyviridae comprise the largest and most important family of RNA plant viruses. An essential overlapping ORF, termed pipo, resides in an internal region of the main polyprotein ORF. Recently, expression of pipo was shown to depend on programmed transcriptional slippage at a conserved GAAAAAA sequence, resulting in the insertion of an extra A into a proportion of viral transcripts, fusing the pipo ORF in frame with the 5' third of the polyprotein ORF. However, the sequence features that mediate slippage have not been characterized. Using a duplicate copy of the pipo slip site region fused into a different genomic location where it can be freely mutated, we investigated the sequence requirements for transcriptional slippage. We find that the leading G is not strictly required, but increased flanking sequence GC content correlates with higher insertion rates. A homopolymeric hexamer is optimal for producing mainly single-nucleotide insertions. We also identify an overabundance of G to A substitutions immediately 3'-adjacent to GAAAAAA in insertion-free transcripts, which we infer to result from a ‘to-fro’ form of slippage during positive-strand synthesis. Analysis of wild-type and reverse complement sequences suggests that slippage occurs preferentially during synthesis of poly(A) and therefore occurs mainly during positive-strand synthesis.

PKC{alpha} and HMGB1 antagonistically control hydrogen peroxide-induced poly-ADP-ribose formation

Mon, 2016-09-19 08:02

Harmful oxidation of proteins, lipids and nucleic acids is observed when reactive oxygen species (ROS) are produced excessively and/or the antioxidant capacity is reduced, causing ‘oxidative stress’. Nuclear poly-ADP-ribose (PAR) formation is thought to be induced in response to oxidative DNA damage and to promote cell death under sustained oxidative stress conditions. However, what exactly triggers PAR induction in response to oxidative stress is incompletely understood. Using reverse phase protein array (RPPA) and in-depth analysis of key stress signaling components, we observed that PAR formation induced by H2O2 was mediated by the PLC/IP3R/Ca2+/PKCα signaling axis. Mechanistically, H2O2-induced PAR formation correlated with Ca2+-dependent DNA damage, which, however, was PKCα-independent. In contrast, PAR formation was completely lost upon knockdown of PKCα, suggesting that DNA damage alone was not sufficient for inducing PAR formation, but required a PKCα-dependent process. Intriguingly, the loss of PAR formation observed upon PKCα depletion was overcome when the chromatin structure-modifying protein HMGB1 was co-depleted with PKCα, suggesting that activation and nuclear translocation of PKCα releases the inhibitory effect of HMGB1 on PAR formation. Together, these results identify PKCα and HMGB1 as important co-regulators involved in H2O2-induced PAR formation, a finding that may have important relevance for oxidative stress-associated pathophysiological conditions.

Defining the essential function of FBP/KSRP proteins: Drosophila Psi interacts with the mediator complex to modulate MYC transcription and tissue growth

Mon, 2016-09-19 08:02

Despite two decades of research, the major function of FBP-family KH domain proteins during animal development remains controversial. The literature is divided between RNA processing and transcriptional functions for these single stranded nucleic acid binding proteins. Using Drosophila, where the three mammalian FBP proteins (FBP1-3) are represented by one ortholog, Psi, we demonstrate the primary developmental role is control of cell and tissue growth. Co-IP-mass spectrometry positioned Psi in an interactome predominantly comprised of RNA Polymerase II (RNA Pol II) transcriptional machinery and we demonstrate Psi is a potent transcriptional activator. The most striking interaction was between Psi and the transcriptional mediator (MED) complex, a known sensor of signaling inputs. Moreover, genetic manipulation of MED activity modified Psi-dependent growth, which suggests Psi interacts with MED to integrate developmental growth signals. Our data suggest the key target of the Psi/MED network in controlling developmentally regulated tissue growth is the transcription factor MYC. As FBP1 has been implicated in controlling expression of the MYC oncogene, we predict interaction between MED and FBP1 might also have implications for cancer initiation and progression.

EZH2 is required for mouse oocyte meiotic maturation by interacting with and stabilizing spindle assembly checkpoint protein BubRI

Mon, 2016-09-19 08:02

Enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 Lys 27 and plays key roles in a variety of biological processes. Stability of spindle assembly checkpoint protein BubR1 is essential for mitosis in somatic cells and for meiosis in oocytes. However, the role of EZH2 in oocyte meiotic maturation was unknown. Here, we presented a mechanism underlying EZH2 control of BubR1 stability in the meiosis of mouse oocytes. We identified a methyltransferase activity-independent function of EZH2 by demonstrating that EZH2 regulates spindle assembly and the polar body I extrusion. EZH2 was increased with the oocyte progression from GVBD to MII, while EZH2 was concentrated on the chromosomes. Interestingly, inhibition of EZH2 methyltranferase activity by DZNep or GSK343 did not affect oocyte meiotic maturation. However, depletion of EZH2 by morpholino led to chromosome misalignment and abnormal spindle assembly. Furthermore, ectopic expression of EZH2 led to oocyte meiotic maturation arrested at the MI stage followed by chromosome misalignment and aneuploidy. Mechanistically, EZH2 directly interacted with and stabilized BubR1, an effect driving EZH2 into the concert of meiosis regulation. Collectively, we provided a paradigm that EZH2 is required for mouse oocyte meiotic maturation.

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment

Mon, 2016-09-19 08:02

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single -H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

A defect in homologous recombination leads to increased translesion synthesis in E. coli

Mon, 2016-09-19 08:02

DNA damage tolerance pathways allow cells to duplicate their genomes despite the presence of replication blocking lesions. Cells possess two major tolerance strategies, namely translesion synthesis (TLS) and homology directed gap repair (HDGR). TLS pathways involve specialized DNA polymerases that are able to synthesize past DNA lesions with an intrinsic risk of causing point mutations. In contrast, HDGR pathways are essentially error-free as they rely on the recovery of missing information from the sister chromatid by RecA-mediated homologous recombination. We have investigated the genetic control of pathway choice between TLS and HDGR in vivo in Escherichia coli. In a strain with wild type RecA activity, the extent of TLS across replication blocking lesions is generally low while HDGR is used extensively. Interestingly, recA alleles that are partially impaired in D-loop formation confer a decrease in HDGR and a concomitant increase in TLS. Thus, partial defect of RecA's capacity to invade the homologous sister chromatid increases the lifetime of the ssDNA.RecA filament, i.e. the ‘SOS signal’. This increase favors TLS by increasing both the TLS polymerase concentration and the lifetime of the TLS substrate, before it becomes sequestered by homologous recombination. In conclusion, the pathway choice between error-prone TLS and error-free HDGR is controlled by the efficiency of homologous recombination.

MutS regulates access of the error-prone DNA polymerase Pol IV to replication sites: a novel mechanism for maintaining replication fidelity

Mon, 2016-09-19 08:02

Translesion DNA polymerases (Pol) function in the bypass of template lesions to relieve stalled replication forks but also display potentially deleterious mutagenic phenotypes that contribute to antibiotic resistance in bacteria and lead to human disease. Effective activity of these enzymes requires association with ring-shaped processivity factors, which dictate their access to sites of DNA synthesis. Here, we show for the first time that the mismatch repair protein MutS plays a role in regulating access of the conserved Y-family Pol IV to replication sites. Our biochemical data reveals that MutS inhibits the interaction of Pol IV with the β clamp processivity factor by competing for binding to the ring. Moreover, the MutS–β clamp association is critical for controlling Pol IV mutagenic replication under normal growth conditions. Thus, our findings reveal important insights into a non-canonical function of MutS in the regulation of a replication activity.

Parallel analysis of ribonucleotide-dependent deletions produced by yeast Top1 in vitro and in vivo

Mon, 2016-09-19 08:02

Ribonucleotides are the most abundant non-canonical component of yeast genomic DNA and their persistence is associated with a distinctive mutation signature characterized by deletion of a single repeat unit from a short tandem repeat. These deletion events are dependent on DNA topoisomerase I (Top1) and are initiated by Top1 incision at the relevant ribonucleotide 3'-phosphodiester. A requirement for the re-ligation activity of Top1 led us to propose a sequential cleavage model for Top1-dependent mutagenesis at ribonucleotides. Here, we test key features of this model via parallel in vitro and in vivo analyses. We find that the distance between two Top1 cleavage sites determines the deletion size and that this distance is inversely related to the deletion frequency. Following the creation of a gap by two Top1 cleavage events, the tandem repeat provides complementarity that promotes realignment to a nick and subsequent Top1-mediated ligation. Complementarity downstream of the gap promotes deletion formation more effectively than does complementarity upstream of the gap, consistent with constraints to realignment of the strand to which Top1 is covalently bound. Our data fortify sequential Top1 cleavage as the mechanism for ribonucleotide-dependent deletions and provide new insight into the component steps of this process.

The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects

Mon, 2016-09-19 08:02

Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes.

Triplex structures induce DNA double strand breaks via replication fork collapse in NER deficient cells

Mon, 2016-09-19 08:02

Structural alterations in DNA can serve as natural impediments to replication fork stability and progression, resulting in DNA damage and genomic instability. Naturally occurring polypurine mirror repeat sequences in the human genome can create endogenous triplex structures evoking a robust DNA damage response. Failures to recognize or adequately process these genomic lesions can result in loss of genomic integrity. Nucleotide excision repair (NER) proteins have been found to play a prominent role in the recognition and repair of triplex structures. We demonstrate using triplex-forming oligonucleotides that chromosomal triplexes perturb DNA replication fork progression, eventually resulting in fork collapse and the induction of double strand breaks (DSBs). We find that cells deficient in the NER damage recognition proteins, XPA and XPC, accumulate more DSBs in response to chromosomal triplex formation than NER-proficient cells. Furthermore, we demonstrate that XPC-deficient cells are particularly prone to replication-associated DSBs in the presence of triplexes. In the absence of XPA or XPC, deleterious consequences of triplex-induced genomic instability may be averted by activating apoptosis via dual phosphorylation of the H2AX protein. Our results reveal that damage recognition by XPC and XPA is critical to maintaining replication fork integrity and preventing replication fork collapse in the presence of triplex structures.

Phosphorylation of Ku70 subunit by cell cycle kinases modulates the replication related function of Ku heterodimer

Mon, 2016-09-19 08:02

The Ku protein, a heterodimer of Ku70 and Ku80, binds to chromosomal replication origins maximally at G1-phase and plays an essential role in assembly of origin recognition complex. However, the mechanism regulating such a critical periodic activity of Ku remained unknown. Here, we establish human Ku70 as a novel target of cyclin B1-Cdk1, which phosphorylates it in a Cy-motif dependent manner. Interestingly, cyclin E1- and A2-Cdk2 also phosphorylate Ku70, and as a result, the protein remains in a phosphorylated state during S-M phases of cell cycle. Intriguingly, the phosphorylation of Ku70 by cyclin-Cdks abolishes the interaction of Ku protein with replication origin due to disruption of the dimer. Furthermore, Ku70 is dephosphorylated in G1-phase, when Ku interacts with replication origin maximally. Strikingly, the over-expression of Ku70 with non-phosphorylable Cdk targets enhances the episomal replication of Ors8 origin and induces rereplication in HeLa cells, substantiating a preventive role of Ku phosphorylation in premature and untimely licensing of replication origin. Therefore, periodic phosphorylation of Ku70 by cyclin-Cdks prevents the interaction of Ku with replication origin after initiation events in S-phase and the dephosphorylation at the end of mitosis facilitates its participation in pre-replication complex formation.

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Fri, 2016-09-02 11:00

A genome-wide approach for detecting novel insertion-deletion variants of mid-range size

Fri, 2016-09-02 11:00

We present SWAN, a statistical framework for robust detection of genomic structural variants in next-generation sequencing data and an analysis of mid-range size insertion and deletions (<10 Kb) for whole genome analysis and DNA mixtures. To identify these mid-range size events, SWAN collectively uses information from read-pair, read-depth and one end mapped reads through statistical likelihoods based on Poisson field models. SWAN also uses soft-clip/split read remapping to supplement the likelihood analysis and determine variant boundaries. The accuracy of SWAN is demonstrated by in silico spike-ins and by identification of known variants in the NA12878 genome. We used SWAN to identify a series of novel set of mid-range insertion/deletion detection that were confirmed by targeted deep re-sequencing. An R package implementation of SWAN is open source and freely available.

Detection and visualization of differential splicing in RNA-Seq data with JunctionSeq

Fri, 2016-09-02 11:00

Although RNA-Seq data provide unprecedented isoform-level expression information, detection of alternative isoform regulation (AIR) remains difficult, particularly when working with an incomplete transcript annotation. We introduce JunctionSeq, a new method that builds on the statistical techniques used by the well-established DEXSeq package to detect differential usage of both exonic regions and splice junctions. In particular, JunctionSeq is capable of detecting differential usage of novel splice junctions without the need for an additional isoform assembly step, greatly improving performance when the available transcript annotation is flawed or incomplete. JunctionSeq also provides a powerful and streamlined visualization toolset that allows bioinformaticians to quickly and intuitively interpret their results. We tested our method on publicly available data from several experiments performed on the rat pineal gland and Toxoplasma gondii, successfully detecting known and previously validated AIR genes in 19 out of 19 gene-level hypothesis tests. Due to its ability to query novel splice sites, JunctionSeq is still able to detect these differences even when all alternative isoforms for these genes were not included in the transcript annotation. JunctionSeq thus provides a powerful method for detecting alternative isoform regulation even with low-quality annotations. An implementation of JunctionSeq is available as an R/Bioconductor package.

Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs

Fri, 2016-09-02 11:00

Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate, using Caenorhabditis elegans, that linear DNAs with short homologies (~35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing.

Multiplexed miRNA northern blots via hybridization chain reaction

Fri, 2016-09-02 11:00

Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2'OMe-RNA probes. The HCR northern blot protocol takes ~1.5 days independent of the number of target RNAs.

Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis

Fri, 2016-09-02 11:00

The sensitive and specific analysis of microRNAs (miRNAs) without using a thermal cycler instrument is significant and would greatly facilitate biological research and disease diagnostics. Although exponential amplification reaction (EXPAR) is the most attractive strategy for the isothermal analysis of miRNAs, its intrinsic limitations of detection efficiency and inevitable non-specific amplification critically restrict its use in analytical sensitivity and specificity. Here, we present a novel asymmetric EXPAR based on a new biotin/toehold featured template. A biotin tag was used to reduce the melting temperature of the primer/template duplex at the 5' terminus of the template, and a toehold exchange structure acted as a filter to suppress the non-specific trigger of EXPAR. The asymmetric EXPAR exhibited great improvements in amplification efficiency and specificity as well as a dramatic extension of dynamic range. The limit of detection for the let-7a analysis was decreased to 6.02 copies (0.01 zmol), and the dynamic range was extended to 10 orders of magnitude. The strategy enabled the sensitive and accurate analysis of let-7a miRNA in human cancer tissues with clearly better precision than both standard EXPAR and RT-qPCR. Asymmetric EXPAR is expected to have an important impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides.

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use

Fri, 2016-09-02 11:00

Genetic decoding is not ‘frozen’ as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational ‘correction’ of problem or ‘savior’ indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression.