Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

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

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

Alignments with non-overlapping moves, inversions and tandem duplications in O ( n 4) time

Sequence alignment is a central problem in bioinformatics. The classical dynamic programming algorithm aligns two sequences by optimizing over possible insertions, deletions and substitutions. However, other evolutionary events can be observed, such as inversions, tandem duplications or moves (transpositions). It has been established that the extension of the problem to move operations is NP-complete. Previous work has shown that an extension restricted to non-overlapping inversions can be solved in O(n 3) with a restricted scoring scheme. In this paper, we show that the alignment problem extended to non-overlapping moves can be solved in O(n 5) for general scoring schemes, O(n 4log n) for concave scoring schemes and O(n 4) for restricted scoring schemes. Furthermore, we show that the alignment problem extended to non-overlapping moves, inversions and tandem duplications can be solved with the same time complexities. Finally, an example of an alignment with non-overlapping moves is provide

ViFi: accurate detection of viral integration and mRNA fusion reveals indiscriminate and unregulated transcription in proximal genomic regions in cervical cancer.

The integration of viral sequences into the host genome is an important driver of tumorigenesis in many viral mediated cancers, notably cervical cancer and hepatocellular carcinoma. We present ViFi, a computational method that combines phylogenetic methods with reference-based read mapping to detect viral integrations. In contrast with read-based reference mapping approaches, ViFi is faster, and shows high precision and sensitivity on both simulated and biological data, even when the integrated virus is a novel strain or highly mutated. We applied ViFi to matched genomic and mRNA data from 68 cervical cancer samples from TCGA and found high concordance between the two. Surprisingly, viral integration resulted in a dramatic transcriptional upregulation in all proximal elements, including LINEs and LTRs that are not normally transcribed. This upregulation is highly correlated with the presence of a viral gene fused with a downstream human element. Moreover, genomic rearrangements suggest the formation of apparent circular extrachromosomal (ecDNA) human-viral structures. Our results suggest the presence of apparent small circular fusion viral/human ecDNA, which correlates with indiscriminate and unregulated expression of proximal genomic elements, potentially contributing to the pathogenesis of HPV-associated cervical cancers. ViFi is available at https://github.com/namphuon/ViFi

A novel bacterial l-arginine sensor controlling c-di-GMP levels in Pseudomonas aeruginosa

Nutrients such as amino acids play key roles in shaping the metabolism of microorganisms in natural environments and in host–pathogen interactions. Beyond taking part to cellular metabolism and to protein synthesis, amino acids are also signaling molecules able to influence group behavior in microorganisms, such as biofilm formation. This lifestyle switch involves complex metabolic reprogramming controlled by local variation of the second messenger 3′, 5′-cyclic diguanylic acid (c-di-GMP). The intracellular levels of this dinucleotide are finely tuned by the opposite activity of dedicated diguanylate cyclases (GGDEF signature) and phosphodiesterases (EAL and HD-GYP signatures), which are usually allosterically controlled by a plethora of environmental and metabolic clues. Among the genes putatively involved in controlling c-di-GMP levels in P. aeruginosa, we found that the multidomain transmembrane protein PA0575, bearing the tandem signature GGDEF-EAL, is an l-arginine sensor able to hydrolyse c-di-GMP. Here, we investigate the basis of arginine recognition by integrating bioinformatics, molecular biophysics and microbiology. Although the role of nutrients such as l-arginine in controlling the cellular fate in P. aeruginosa (including biofilm, pathogenicity and virulence) is already well established, we identified the first l-arginine sensor able to link environment sensing, c-di-GMP signaling and biofilm formation in this bacterium