R-Loops as Cellular Regulators and Genomic Threats.

R Loops: From Physiological to Pathological Roles

Oncogene activation disturbs cellular processes and accommodates a complex landscape of changes in the genome that contribute to genomic instability, which accelerates mutation rates and promotes tumorigenesis. Part of this cellular turmoil involves deregulation of physiologic DNA replication, widely described as replication stress. Oncogene-induced replication stress is an early driver of genomic instability and is attributed to a plethora of factors, most notably aberrant origin firing, replication-transcription collisions, reactive oxygen species, and defective nucleotide metabolism.Significance: Replication stress is a fundamental step and an early driver of tumorigenesis and has been associated with many activated oncogenes. Deciphering the mechanisms that contribute to the replication stress response may provide new avenues for targeted cancer treatment. In this review, we discuss the latest findings on the DNA replication stress response and examine the various mechanisms through which activated oncogenes induce replication stress. Cancer Discov; 8(5); 537-55. ©2018 AACR.

https://cancerdiscovery.aacrjournals.org/content/candisc/8/5/537.full.pdf

Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into Place.

R-loops are three-stranded structures that harbour an RNA-DNA hybrid and frequently form during transcription. R-loop misregulation is associated with DNA damage, transcription elongation defects, hyper-recombination and genome instability. In contrast to such 'unscheduled' R-loops, evidence is mounting that cells harness the presence of RNA-DNA hybrids in scheduled, 'regulatory' R-loops to promote DNA transactions, including transcription termination and other steps of gene regulation, telomere stability and DNA repair. R-loops formed by cellular RNAs can regulate histone post-translational modification and may be recognized by dedicated reader proteins. The two-faced nature of R-loops implies that their formation, location and timely removal must be tightly regulated. In this Perspective, we discuss the cellular processes that regulatory R-loops modulate, the regulation of R-loops and the potential differences that may exist between regulatory R-loops and unscheduled R-loops.

Regulatory R-loops as facilitators of gene expression and genome stability.

The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with 'ready-to-use' deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and that may proliferate in public database repositories affecting all downstream analyses. As a case study, we provide examples of the Atlantic cod genome, whose sequencing and assembly were hindered by a particularly high prevalence of tandem repeats. We complement this case study with examples from other species, where mis-annotations and sequencing errors have propagated into protein databases. With this review, we aim to raise the awareness level within the community of database users, and alert scientists working in the underlying workflow of database creation that the data they omit or improperly assemble may well contain important biological information valuable to others.

/pdf/tandem-repeats-lead-to-sequence-assembly-errors-and-impose-37tmyr6uto.pdf

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases

Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis

Substitutions Are Boring: Some Arguments about Parallel Mutations and High Mutation Rates.

Short tandem repeat (STR) mutations may comprise more than half of the mutations in eukaryotic coding DNA, yet STR variation is rarely examined as a contributor to complex traits. We assessed this contribution across a collection of 96 strains of Arabidopsis thaliana, genotyping 2046 STR loci each, using highly parallel STR sequencing with molecular inversion probes. We found that 95% of examined STRs are polymorphic, with a median of six alleles per STR across these strains. STR expansions (large copy number increases) are found in most strains, several of which have evident functional effects. These include three of six intronic STR expansions we found to be associated with intron retention. Coding STRs were depleted of variation relative to noncoding STRs, and we detected a total of 56 coding STRs (11%) showing low variation consistent with the action of purifying selection. In contrast, some STRs show hypervariable patterns consistent with diversifying selection. Finally, we detected 133 novel STR-phenotype associations under stringent criteria, most of which could not be detected with SNPs alone, and validated some with follow-up experiments. Our results support the conclusion that STRs constitute a large, unascertained reservoir of functionally relevant genomic variation.

/pdf/massive-variation-of-short-tandem-repeats-with-functional-3w2hj7x85r.pdf

Massive variation of short tandem repeats with functional consequences across strains of Arabidopsis thaliana.

The bacterium Bacillus subtilis is capable of two kinds of flagellum-mediated motility: swimming, which occurs in liquid, and swarming, which occurs on a surface. Swarming is distinct from swimming in that it requires secretion of a surfactant, an increase in flagellar density, and perhaps additional factors. Here we report a new gene, swrD, located within the 32 gene fla-che operon dedicated to flagellar biosynthesis and chemotaxis, which when mutated abolished swarming motility. SwrD was not required for surfactant production, flagellar gene expression, or an increase in flagellar number. Instead, SwrD was required to increase flagellar power. Mutation of swrD reduced swimming speed and torque of tethered flagella, and all swrD-related phenotypes were restored when the stator subunits MotA and MotB were overexpressed either by spontaneous suppressor mutations or by artificial induction. We conclude that swarming motility requires flagellar power in excess of that which is needed to swim.IMPORTANCE Bacteria swim in liquid and swarm over surfaces by rotating flagella, but the difference between swimming and swarming is poorly understood. Here we report that SwrD of Bacillus subtilis is necessary for swarming because it increases flagellar torque and cells mutated for swrD swim with reduced speed. How flagellar motors generate power is primarily studied in Escherichia coli, and SwrD likely increases power in other organisms, like the Firmicutes, Clostridia, Spirochaetes, and the Deltaproteobacteria.

SwrD (YlzI) Promotes Swarming in Bacillus subtilis by Increasing Power to Flagellar Motors.

The ribosomal RNA genes (rDNA) are tandemly arrayed in most eukaryotes and exhibit vast copy number variation. There is growing interest in integrating this variation into genotype-phenotype associations. Here, we explored a possible association of rDNA copy number variation with autism spectrum disorder and found no difference between probands and unaffected siblings. Because short-read sequencing estimates of rDNA copy number are error prone, we sought to validate our 45S estimates. Previous studies reported tightly correlated, concerted copy number variation between the 45S and 5S arrays, which should enable the validation of 45S copy number estimates with pulsed-field gel-verified 5S copy numbers. Here, we show that the previously reported strong concerted copy number variation may be an artifact of variable data quality in the earlier published 1000 Genomes Project sequences. We failed to detect a meaningful correlation between 45S and 5S copy numbers in thousands of samples from the high-coverage Simons Simplex Collection dataset as well as in the recent high-coverage 1000 Genomes Project sequences. Our findings illustrate the challenge of genotyping repetitive DNA regions accurately and call into question the accuracy of recently published studies of rDNA copy number variation in cancer that relied on diverse publicly available resources for sequence data.

/pdf/thousands-of-high-quality-sequencing-samples-fail-to-show-1h05zxwkwq.pdf

Ashley N. Hall

Papers

Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis

Substitutions Are Boring: Some Arguments about Parallel Mutations and High Mutation Rates.

Massive variation of short tandem repeats with functional consequences across strains of Arabidopsis thaliana.

SwrD (YlzI) Promotes Swarming in Bacillus subtilis by Increasing Power to Flagellar Motors.

Thousands of high-quality sequencing samples fail to show meaningful correlation between 5S and 45S ribosomal DNA arrays in humans.