TGT damages its substrate tRNAs by the formation of abasic sites in the anticodon loop
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB82780 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR14376600.fastq.gz | Fastqsanger.gz | |||
| ERR14376601.fastq.gz | Fastqsanger.gz | |||
| ERR14376602.fastq.gz | Fastqsanger.gz | |||
| ERR14376603.fastq.gz | Fastqsanger.gz | |||
| ERR14376604.fastq.gz | Fastqsanger.gz |
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Project description:Abasic (AP) sites are one of the most common forms of DNA damage which can lead to polymerase stalling, strand breaks and mutations. We developed snA-seq, a mapping method that reveals the location of abasic sites at base-resolution. Using synthetic DNA, we show that high selectivity for AP DNA is achieved. We use this method to explore the distribution of thymine modifications in the Leishmania major genome, by converting these into abasic sites using a glycosylase enzyme. We also apply snAP-seq to the human genome to study the distribution of endogenous AP sites, in both APE1 knockdown and control cells.
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Project description:The AP-seq provides a genome-wide method for detecting abasic sites among the human and mouse genomes Purpose: Abasic sites are one of the most frequent DNA damage among the genome , which often carries harmful consequences for the genome stability and the normal cell functions. So it is necessary to develop a whole genome abasic sites (AP sites) profiling method to detect all the AP sites among the genome, which we called the AP-seq. Methods: The human and mouse genome are processed with enzymatic fragmentation, damage repair, biotin-dU labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: The reproducibility of our two biological replicates shows a high correlation which larger than 0.95. And the overlap AP site peaks between the HEK293T and MCF-7 cell line are over 50%, which show a great similarity between the different cell lines. Totally, we identified more than 2000 and 1000 abasic site peaks in human, and mouse cell lines separately. Moreover, the distribution of these AP sites prefer to locate in the intergenic regions and introns.
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Project description:Transfer RNAs (tRNAs) in bacteriophage genomes are widespread across bacterial host genera, but their exact function has remained unclear for more than 50 years. Several hypotheses have been proposed, and the most widely accepted one is codon compensation, which suggests that phages encode tRNAs that supplement codons that are less frequently used by the host. Here, we combine several observations and propose a new hypothesis that phage-encoded tRNAs counteract the tRNA-depleting strategies of the host using enzymes such as VapC, PrrC, Colicin D, and Colicin E5 to defend from viral infection. Based on mutational patterns of anticodon loops of tRNAs encoded by phages, we predict that these tRNAs are insensitive to host tRNAses. For phage-encoded tRNAs targeted in the anticodon itself, we observe that phages typically avoid encoding these tRNAs, further supporting the hypothesis that phage tRNAs are selected to be insensitive to host anticodon nucleases. Altogether, our results support the hypothesis that phage-encoded tRNAs have evolved to be insensitive to host anticodon nucleases.
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Project description:During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem-loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASL(CCCG) and ASL(ACCC) (5'-3' nomenclature) form unpredicted anticodon-codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASL(ACGA) bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon-codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.
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Project description:Single-nucleotide resolution mapping of DNA abasic sites by snAP-seq
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Project description:The CCA-adding enzyme adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. Here, we report deep sequencing analysis of the 3' ends of tRNA-Ser-CGA and tRNA-Ser-UGA from S. cerevisiae strains and show that hypomodified mature tRNAs are subjected to CCACCA (or poly(A) addition) as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addtion is a universal mechanism for controlling tRNA levels and preventing errors in translation. 121 samples analyzed in total, representing time courses of 10 different yeast strains; Biological replicates for each time point are included
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Project description:DNA damage plays critical role in biology and disease, however, precise understanding of how different types of DNA damage affect cellular functions is far from clear. A major underlying reason for this is paucity of high-resolution methods that can map locations of different types of DNA damage in complex genomes such as those of mammals. Here, we present development and validation of SSiNGLe-AP method that can map a common type of DNA damage, abasic (AP) sites, genome-wide and with high-resolution. We applied this method to six different tissues of mice of different ages and human cancer cell lines. We found non-random distribution of AP sites in mammalian genome that exhibits dynamic enrichment at specific genomic locations, and is significantly influenced by gene expression, age and especially by tissue-type. Overall, these results suggest that we are at the very beginning of understanding the true complexities of genomic patterns of DNA damage.
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