Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage.

Initiation of chromosome replication in bacteria is precisely timed in the cell cycle. Bacteria that harbor multiple chromosomes face the additional challenge of orchestrating replication initiation of different chromosomes. In Vibrio cholerae, the smaller of its two chromosomes, Chr2, initiates replication after Chr1 such that both chromosomes terminate replication synchronously. The delay is due to the dependence of Chr2 initiation on the replication of a site, crtS, on Chr1. The mechanism by which replication of crtS allows Chr2 replication remains unclear. Here, we show that blocking Chr1 replication indeed blocks Chr2 replication, but providing an extra crtS copy in replication-blocked Chr1 permitted Chr2 replication. This demonstrates that unreplicated crtS copies have significant activity, and suggests that a role of replication is to double the copy number of the site that sufficiently increases its activity for licensing Chr2 replication. We further show that crtS activity promotes the Chr2-specific initiator function and that this activity is required in every cell cycle, as would be expected of a cell-cycle regulator. This study reveals how increase of gene dosage through replication can be utilized in a critical regulatory switch.

Enhanced eMAGE applied to identify genetic factors of nuclear hormone receptor dysfunction via combinatorial gene editing.

Technologies that generate precise combinatorial genome modifications are well suited to dissect the polygenic basis of complex phenotypes and engineer synthetic genomes. Genome modifications with engineered nucleases can lead to undesirable repair outcomes through imprecise homology-directed repair, requiring non-cleavable gene editing strategies. Eukaryotic multiplex genome engineering (eMAGE) generates precise combinatorial genome modifications in Saccharomyces cerevisiae without generating DNA breaks or using engineered nucleases. Here, we systematically optimize eMAGE to achieve 90% editing frequency, reduce workflow time, and extend editing distance to 20 kb. We further engineer an inducible dominant negative mismatch repair system, allowing for high-efficiency editing via eMAGE while suppressing the elevated background mutation rate 17-fold resulting from mismatch repair inactivation. We apply these advances to construct a library of cancer-associated mutations in the ligand-binding domains of human estrogen receptor alpha and progesterone receptor to understand their impact on ligand-independent autoactivation. We validate that this yeast model captures autoactivation mutations characterized in human breast cancer models and further leads to the discovery of several previously uncharacterized autoactivating mutations. This work demonstrates the development and optimization of a cleavage-free method of genome editing well suited for applications requiring efficient multiplex editing with minimal background mutations.

Recombineering and MAGE.

Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes. Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE). MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies. First described in Escherichia coli, both MAGE and recombineering are being rapidly translated into diverse prokaryotes and even into eukaryotic cells. Together, this modern set of tools offers the promise of radically improving the scope and throughput of experimental biology by providing powerful new methods to ease the genetic manipulation of model and non-model organisms. In this Primer, we describe recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools. We then look forward to the future of genetic engineering.

A Requirement for Global Transcription Factor Lrp in Licensing Replication of Vibrio cholerae Chromosome 2.

The human pathogen, Vibrio cholerae, belongs to the 10% of bacteria in which the genome is divided. Each of its two chromosomes, like bacterial chromosomes in general, replicates from a unique origin at fixed times in the cell cycle. Chr1 initiates first, and upon duplication of a site in Chr1, crtS, Chr2 replication initiates. Recent in vivo experiments demonstrate that crtS binds the Chr2-specific initiator RctB and promotes its initiator activity by remodeling it. Compared to the well-defined RctB binding sites in the Chr2 origin, crtS is an order of magnitude longer, suggesting that other factors can bind to it. We developed an in vivo screen to identify additional crtS-binding proteins and identified the global transcription factor, Lrp, as one such protein. Studies in vivo and in vitro indicate that Lrp binds to crtS and facilitates RctB binding to crtS. Chr2 replication is severely defective in the absence of Lrp, indicative of a critical role of the transcription factor in licensing Chr2 replication. Since Lrp responds to stresses such as nutrient limitation, its interaction with RctB presumably sensitizes Chr2 replication to the physiological state of the cell.