Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease

The BcgI endonuclease exemplifies a subset of restriction enzymes, the Type IIB class, which make two double-strand breaks (DSBs) at each copy of their recognition sequence, one either side of the site, to excise the sequence from the remainder of the DNA. In this study, we show that BcgI is essentially inactive when bound to a single site and that to cleave a DNA with one copy of its recognition sequence, it has to act in trans, bridging two separate DNA molecules. We also show that BcgI makes the two DSBs at an individual site in a highly concerted manner. Intermediates cut on one side of the site do not accumulate during the course of the reaction: instead, the DNA is converted straight to the final products cut on both sides. On DNA with two sites, BcgI bridges the sites in cis and then generally proceeds to cut both strands on both sides of both sites without leaving the DNA. The BcgI restriction enzyme can thus excise two DNA segments together, by cleaving eight phosphodiester bonds within a single-DNA binding event

Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways

High-throughput genomics and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries and synthetic pathways. Here, we describe the development of a novel and extremely simple cloning method, circular polymerase extension cloning (CPEC). This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recombination is required. In this study, we elucidate the CPEC reaction mechanism and demonstrate its usage in demanding synthetic biology applications such as one-step assembly and cloning of complex combinatorial libraries and multi-component pathways

The structure of SgrAI bound to DNA; recognition of an 8 base pair target

The three-dimensional X-ray crystal structure of the ‘rare cutting’ type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca2+ bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn2+, with strong binding at the protein–DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI

Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases

Zinc finger nucleases (ZFNs) facilitate tailor-made genomic modifications in vivo through the creation of targeted double-stranded breaks. They have been employed to modify the genomes of plants and animals, and cell-based therapies utilizing ZFNs are undergoing clinical trials. However, many ZFNs display dose-dependent toxicity presumably due to the generation of undesired double-stranded breaks at off-target sites. To evaluate the parameters influencing the functional specificity of ZFNs, we compared the in vivo activity of ZFN variants targeting the zebrafish kdrl locus, which display both high on-target activity and dose-dependent toxicity. We evaluated their functional specificity by assessing lesion frequency at 141 potential off-target sites using Illumina sequencing. Only a minority of these off-target sites accumulated lesions, where the thermodynamics of zinc finger–DNA recognition appear to be a defining feature of active sites. Surprisingly, we observed that both the specificity of the incorporated zinc fingers and the choice of the engineered nuclease domain could independently influence the fidelity of these ZFNs. The results of this study have implications for the assessment of likely off-target sites within a genome and point to both zinc finger-dependent and -independent characteristics that can be tailored to create ZFNs with greater precision

A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors

DNA looping by FokI: the impact of synapse geometry on loop topology at varied site orientations

Most restriction endonucleases, including FokI, interact with two copies of their recognition sequence before cutting DNA. On DNA with two sites they act in cis looping out the intervening DNA. While many restriction enzymes operate symmetrically at palindromic sites, FokI acts asymmetrically at a non-palindromic site. The directionality of its sequence means that two FokI sites can be bridged in either parallel or anti-parallel alignments. Here we show by biochemical and single-molecule biophysical methods that FokI aligns two recognition sites on separate DNA molecules in parallel and that the parallel arrangement holds for sites in the same DNA regardless of whether they are in inverted or repeated orientations. The parallel arrangement dictates the topology of the loop trapped between sites in cis: the loop from inverted sites has a simple 180° bend, while that with repeated sites has a convoluted 360° turn. The ability of FokI to act at asymmetric sites thus enabled us to identify the synapse geometry for sites in trans and in cis, which in turn revealed the relationship between synapse geometry and loop topology

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes

The Heme Biosynthetic Pathway of the Obligate Wolbachia Endosymbiont of Brugia malayi as a Potential Anti-filarial Drug Target

Human filarial nematodes are causative agents of elephantiasis and African river blindness, which are among the most debilitating tropical diseases. Currently used drugs mainly affect microfilariae (mf) and have less effect on adult filarial nematodes, which can live in the human host for more than a decade. Filariasis drug control strategy relies on recurrent mass drug administration for many years. Development of novel drugs is also urgently needed due to the threat of drug resistance occurrence. Most filarial worms harbor an obligate endosymbiotic bacterium, Wolbachia, whose presence has been identified as a potential drug target. Comparative genomics had suggested Wolbachia heme biosynthesis as a potential drug target, and we present an analysis of selected enzymes alongside their human homologues from several different aspects—gene phylogenetic analyses, in vitro enzyme kinetic and inhibition assays and heme-deficient E. coli complementation assays. We also conducted ex vivo Brugia malayi viability assays using heme pathway inhibitors. These experiments demonstrate that heme biosynthesis could be critical for filarial worm survival and thus is a potential anti-filarial drug target set