Replication Stress-Induced Chromosome Breakage Is Correlated with Replication Fork Progression and Is Preceded by Single-Stranded DNA Formation

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or “collapsed” replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay

ABSTRACT Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro . The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate

Fragile Genomic Sites Are Associated with Origins of Replication

Genome rearrangements are mediators of evolution and disease. Such rearrangements are frequently bounded by transfer RNAs (tRNAs), transposable elements, and other repeated elements, suggesting a functional role for these elements in creating or repairing breakpoints. Though not well explored, there is evidence that origins of replication also colocalize with breakpoints. To investigate a potential correlation between breakpoints and origins, we analyzed evolutionary breakpoints defined between Saccharomyces cerevisiae and Kluyveromyces waltii and S. cerevisiae and a hypothetical ancestor of both yeasts, as well as breakpoints reported in the experimental literature. We find that origins correlate strongly with both evolutionary breakpoints and those described in the literature. Specifically, we find that origins firing earlier in S phase are more strongly correlated with breakpoints than are later-firing origins. Despite origins being located in genomic regions also bearing tRNAs and Ty elements, the correlation we observe between origins and breakpoints appears to be independent of these genomic features. This study lays the groundwork for understanding the mechanisms by which origins of replication may impact genome architecture and disease

Microbial stimulation of oxytocin release from the intestinal epithelium via secretin signaling

ABSTRACTIntestinal microbes impact the health of the intestine and organs distal to the gut. Limosilactobacillus reuteri is a human intestinal microbe that promotes normal gut transit, the anti-inflammatory immune system, wound healing, normal social behavior in mice, and prevents bone reabsorption. Oxytocin impacts these functions and oxytocin signaling is required for L. reuteri-mediated wound healing and social behavior; however, the events in the gut leading to oxytocin stimulation and beneficial effects are unknown. Here we report evolutionarily conserved oxytocin production in the intestinal epithelium through analysis of single-cell RNA-Seq datasets and imaging of human and mouse intestinal tissues. Moreover, human intestinal organoids produce oxytocin, demonstrating that the intestinal epithelium is sufficient to produce oxytocin. We find that L. reuteri facilitates oxytocin secretion from human intestinal tissue and human intestinal organoids. Finally, we demonstrate that stimulation of oxytocin secretion by L. reuteri is dependent on the gut hormone secretin, which is produced in enteroendocrine cells, while oxytocin itself is produced in enterocytes. Altogether, this work demonstrates that oxytocin is produced and secreted from enterocytes in the intestinal epithelium in response to secretin stimulated by L. reuteri. This work thereby identifies oxytocin as an intestinal hormone and provides mechanistic insight into avenues by which gut microbes promote host health

Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification.

DNA replication errors are a major driver of evolution--from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model--Origin-Dependent Inverted-Repeat Amplification (ODIRA)-proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error-the ligation of leading and lagging nascent strands to create "closed" forks-can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent--a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution

The human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate phylum sibling to Cyanobacteria.

Cyanobacteria were responsible for the oxygenation of the ancient atmosphere; however, the evolution of this phylum is enigmatic, as relatives have not been characterized. Here we use whole genome reconstruction of human fecal and subsurface aquifer metagenomic samples to obtain complete genomes for members of a new candidate phylum sibling to Cyanobacteria, for which we propose the designation 'Melainabacteria'. Metabolic analysis suggests that the ancestors to both lineages were non-photosynthetic, anaerobic, motile, and obligately fermentative. Cyanobacterial light sensing may have been facilitated by regulators present in the ancestor of these lineages. The subsurface organism has the capacity for nitrogen fixation using a nitrogenase distinct from that in Cyanobacteria, suggesting nitrogen fixation evolved separately in the two lineages. We hypothesize that Cyanobacteria split from Melainabacteria prior or due to the acquisition of oxygenic photosynthesis. Melainabacteria remained in anoxic zones and differentiated by niche adaptation, including for symbiosis in the mammalian gut. DOI:http://dx.doi.org/10.7554/eLife.01102.001

An <i>in vitro</i> construct to test for fork reversal and cross-strand ligation.

<p>An artificial replication fork was generated using commercially synthesized oligonucleotides (sizes of 100 and 99 nucleotides, with a 5’-phosphate on the 99 nucleotide lagging oligonucleotide). (A) To facilitate assembly of the fork from its component strands, the “parental” and “nascent” strands of the leading arm of the duplex, and likewise, the two strands of the lagging arm of the replication fork were covalently joined by a short linker. Complementary sequences in the two, 30 base, single stranded tails allow the fork to assemble <i>in vitro</i>. Features engineered into the two oligos include a 7 bp inverted repeat (blue sequences highlighted with small arrows) separated by 11 nucleotides of non-complementary sequence, and various restriction sites to facilitate evaluation of the fork and its products. (B) The single stranded stretch on the lagging strand exposes two complementary copies of the inverted repeat (in blue with arrow) which are expected to fold into a stem-loop structure to achieve the lowest free energy state for the forked molecule. (C and D) Breathing of the duplexes at the fork facilitates fork reversal and generates alternative conformations by branch migration—all with identical numbers of paired bases. The structures in B and D reflect the two extreme branch-migrated forms, with C representing only one of 25 possible intermediates. The two nucleotides in red are brought into proximity in the branch-migrated forms, providing a substrate for ligation by DNA ligase. Completion of the ligation step creates an <i>Xho</i>I restriction site. PCR primers (shown in C) target the two linkers.</p