Thinking Outside the Cell: Replicating Replication In Vitro.

Benchmark studies by Yeeles et al. (2017), Kurat et al. (2017), and Devbhandari et al. (2017) have reconstituted rapid and regulated budding yeast DNA replication on naked and chromatinized templates in vitro, providing key insights into this fundamentally important process.

Corrigendum: Commensal bacteria make GPCR ligands that mimic human signalling molecules.

This corrects the article DOI: 10.1038/nature23874.

Commensal bacteria make GPCR ligands that mimic human signalling molecules.

Commensal bacteria are believed to have important roles in human health. The mechanisms by which they affect mammalian physiology remain poorly understood, but bacterial metabolites are likely to be key components of host interactions. Here we use bioinformatics and synthetic biology to mine the human microbiota for N-acyl amides that interact with G-protein-coupled receptors (GPCRs). We found that N-acyl amide synthase genes are enriched in gastrointestinal bacteria and the lipids that they encode interact with GPCRs that regulate gastrointestinal tract physiology. Mouse and cell-based models demonstrate that commensal GPR119 agonists regulate metabolic hormones and glucose homeostasis as efficiently as human ligands, although future studies are needed to define their potential physiological role in humans. Our results suggest that chemical mimicry of eukaryotic signalling molecules may be common among commensal bacteria and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a possible small-molecule therapeutic modality (microbiome-biosynthetic gene therapy).

A novel single alpha-helix DNA-binding domain in CAF-1 promotes gene silencing and DNA damage survival through tetrasome-length DNA selectivity and spacer function.

The histone chaperone chromatin assembly factor 1 (CAF-1) deposits two nascent histone H3/H4 dimers onto newly replicated DNA forming the central core of the nucleosome known as the tetrasome. How CAF-1 ensures there is sufficient space for the assembly of tetrasomes remains unknown. Structural and biophysical characterization of the lysine/glutamic acid/arginine-rich (KER) region of CAF-1 revealed a 128-Å single alpha-helix (SAH) motif with unprecedented DNA-binding properties. Distinct KER sequence features and length of the SAH drive the selectivity of CAF-1 for tetrasome-length DNA and facilitate function in budding yeast. In vivo, the KER cooperates with the DNA-binding winged helix domain in CAF-1 to overcome DNA damage sensitivity and maintain silencing of gene expression. We propose that the KER SAH links functional domains within CAF-1 with structural precision, acting as a DNA-binding spacer element during chromatin assembly.

A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9.

Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome.