Nucleic Acids Research最新文献

DNA-protein crosslinks (DPCs) challenge faithful DNA replication and smooth passage of genomic information. Our study unveils the cullin E3 ubiquitin ligase Rtt101 as a DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types including topoisomerase 1 crosslinks, in the same pathway as the ubiquitin-dependent aspartic protease Ddi1. Using an in vivo inducible Top1-mimicking DPC system, we reveal the significant impact of Rtt101 ubiquitination on DPC removal across different cell cycle phases. High-throughput methods coupled with next-generation sequencing specifically highlight the association of Rtt101 with replisomes as well as colocalization with DPCs. Our findings establish Rtt101 as a main contributor to DPC repair throughout the yeast cell cycle.

DNA damage has been implicated in the stimulation of the type 1 interferon (T1IFN) response. Here, we show that downregulation of the DNA repair protein, polynucleotide kinase/phosphatase (PNKP), in a variety of cell lines causes robust phosphorylation of STAT1, upregulation of interferon-stimulated genes and persistent accumulation of cytosolic DNA, all of which are indicators for the activation of the T1IFN response. Furthermore, this did not require damage induction by ionizing radiation. Instead, our data revealed that production of reactive oxygen species (ROS) synergises with PNKP loss to potentiate the T1IFN response, and that loss of PNKP significantly compromises mitochondrial DNA (mtDNA) integrity. Depletion of mtDNA or treatment of PNKP-depleted cells with ROS scavengers abrogated the T1IFN response, implicating mtDNA as a significant source of the cytosolic DNA required to potentiate the T1IFN response. The STING signalling pathway is responsible for the observed increase in the pro-inflammatory gene signature in PNKP-depleted cells. While the response was dependent on ZBP1, cGAS only contributed to the response in some cell lines. Our data have implications for cancer therapy, since PNKP inhibitors would have the potential to stimulate the immune response, and also to the neurological disorders associated with PNKP mutation.

The endonuclease activity of Pms1 directs mismatch repair by generating a nick in the newly replicated DNA strand. Inactivating Pms2, the human homologue of yeast Pms1, increases the chances of colorectal and uterine cancers. Here we use whole genome sequencing to show that loss of this endonuclease activity, via the pms1-DE variant, results in strong mutator effects throughout the Saccharomyces cerevisiae genome. Mutation rates are strongly increased for mutations resulting from all types of single-base substitutions and for a wide variety of single- and multi-base indel mutations. Rates for these events are further increased in strains combining pms1-DE with mutator variants of each of the three major leading and lagging strand replicases. In all cases, mutation rates, spectra, biases, and context preferences are statistically indistinguishable from strains with equivalent polymerases but lacking initial mismatch recognition due to deletion of MSH2. This implies that, across the nuclear genome, strand discrimination via the Pms1 endonuclease is as important for MMR as is initial mismatch recognition by Msh2 heterodimers.

Recently, we introduced a chromatin immunoprecipitation (ChIP) technique utilizing the human DNA Fragmentation Factor (DFF) to digest the DNA prior to immunoprecipitation (DFF-ChIP) that provides the precise location of transcription complexes and their interactions with neighboring nucleosomes. Here we expand the technique to new targets and provide useful information concerning purification of DFF, digestion conditions, and the impact of crosslinking. DFF-ChIP analysis was performed individually for subunits of Mediator, DSIF, and NELF that that do not interact with DNA directly, but rather interact with RNA polymerase II (Pol II). We found that Mediator was associated almost exclusively with preinitiation complexes (PICs). DSIF and NELF were associated with engaged Pol II and, in addition, potential intermediates between PICs and early initiation complexes. DFF-ChIP was then used to analyze the occupancy of a tight binding transcription factor, CTCF, and a much weaker binding factor, glucocorticoid receptor (GR), with and without crosslinking. These results were compared to those from standard ChIP-Seq that employs sonication and to CUT&RUN which utilizes MNase to fragment the genomic DNA. Our findings indicate that DFF-ChIP reveals details of occupancy that are not available using other methods including information revealing pertinent protein:protein interactions.

Long terminal repeat (LTR)-retrotransposons are significant contributors to the evolution and diversity of eukaryotic genomes. Their RNA genomes (gRNA) serve as a template for protein synthesis and reverse transcription to a DNA copy, which can integrate into the host genome. Here, we used the SHAPE-MaP strategy to explore Ty3 retrotransposon gRNA structure in yeast and under cell-free conditions. Our study reveals the structural dynamics of Ty3 gRNA and the well-folded core, formed independently of the cellular environment. Based on the detailed map of Ty3 gRNA structure, we characterized the structural context of cis-acting sequences involved in reverse transcription and frameshifting. We also identified a novel functional sequence as a potential initiator for Ty3 gRNA dimerization. Our data indicate that the dimer is maintained by direct interaction between short palindromic sequences at the 5' ends of the two Ty3 gRNAs, resembling the model characteristic for other retroelements like HIV-1 and Ty1. This work points out a range of cell-dependent and -independent Ty3 gRNA structural changes that provide a solid background for studies on RNA structure-function relationships important for retroelement biology.

Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes.

Non-segmented negative-strand (NNS) RNA viruses, such as rabies, Nipah and Ebola, produce 5'-capped and 3'-polyadenylated mRNAs resembling higher eukaryotic mRNAs. Here, we developed a transcription elongation-coupled pre-mRNA capping system for vesicular stomatitis virus (VSV, a prototypic NNS RNA virus). Using this system, we demonstrate that the single-polypeptide RNA-dependent RNA polymerase (RdRp) large protein (L) catalyzes all pre-mRNA modifications co-transcriptionally in the following order: (i) 5'-capping (polyribonucleotidylation of GDP) to form a GpppA cap core structure, (ii) 2'-O-methylation of GpppA into GpppAm, (iii) guanine-N7-methylation of GpppAm into m7GpppAm (cap 1), (iv) 3'-polyadenylation to yield a poly(A) tail. The GDP polyribonucleotidyltransferase (PRNTase) domain of L generated capped pre-mRNAs of 18 nucleotides or longer via the formation of covalent enzyme-pre-mRNA intermediates. The single methyltransferase domain of L sequentially methylated the cap structure only when pre-mRNAs of 40 nucleotides or longer were associated with elongation complexes. These results suggest that the formation of pre-mRNA closed loop structures in elongation complexes via the RdRp and PRNTase domains followed by the RdRp and MTase domains on the same polypeptide is required for the cap 1 formation during transcription. Taken together, our findings indicate that NNS RNA virus L acts as an all-in-one viral mRNA assembly machinery.

Our genome is exposed to a wide variety of DNA-damaging agents. If left unrepaired, this damage can be converted into mutations that promote carcinogenesis or the development of genetically inherited diseases. As a result, researchers and clinicians require tools that can detect DNA damage and mutations with exceptional sensitivity. In this study, we describe a massively parallel sequencing tool termed Mutation And DNA Damage Detection-seq (MADDD-seq) that is capable of detecting O6-methyl guanine lesions and mutations simultaneously, with a single assay. To illustrate the dual capabilities of MADDD-seq, we treated WT and DNA repair deficient yeast cells with the DNA-damaging agent MNNG and tracked DNA lesions and mutations over a 24-h time period. This approach allowed us to identify thousands of DNA adducts and mutations in a single sequencing run and gain deep insight into the kinetics of DNA repair and mutagenesis.

The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during the translation of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded proteins synthesized by mitochondrial ribosomes also contain polyproline stretches, an EF-P/eIF5A mitochondrial counterpart remains unidentified. Here, we show that the missing factor is TACO1, a protein causative of a juvenile form of neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be a translational activator of COX1 mRNA. By using a combination of metabolic labeling, puromycin release and mitoribosome profiling experiments, we show that TACO1 is required for the rapid synthesis of the polyproline-rich COX1 and COX3 cytochrome c oxidase subunits, while its requirement is negligible for other mitochondrial DNA-encoded proteins. In agreement with a role in translation efficiency regulation, we show that TACO1 cooperates with the N-terminal extension of the large ribosomal subunit bL27m to provide stability to the peptidyl-transferase center during elongation. This study illuminates the translation elongation dynamics within human mitochondria, a TACO1-mediated biological mechanism in place to mitigate mitoribosome stalling at polyproline stretches during protein synthesis, and the pathological implications of its malfunction.

Somatic structural variations (SVs) in cancer can shuffle DNA content in the genome, relocate regulatory elements, and alter genome organization. Enhancer hijacking occurs when SVs relocate distal enhancers to activate proto-oncogenes. However, most enhancer hijacking studies have only focused on protein-coding genes. Here, we develop a computational algorithm 'HYENA' to identify candidate oncogenes (both protein-coding and non-coding) activated by enhancer hijacking based on tumor whole-genome and transcriptome sequencing data. HYENA detects genes whose elevated expression is associated with somatic SVs by using a rank-based regression model. We systematically analyze 1146 tumors across 25 types of adult tumors and identify a total of 108 candidate oncogenes including many non-coding genes. A long non-coding RNA TOB1-AS1 is activated by various types of SVs in 10% of pancreatic cancers through altered 3-dimensional genome structure. We find that high expression of TOB1-AS1 can promote cell invasion and metastasis. Our study highlights the contribution of genetic alterations in non-coding regions to tumorigenesis and tumor progression.