DNA looping by FokI: the impact of twisting and bending rigidity on protein-induced looping dynamics

Protein-induced DNA looping is crucial for many genetic processes such as transcription, gene regulation and DNA replication. Here, we use tethered-particle motion to examine the impact of DNA bending and twisting rigidity on loop capture and release, using the restriction endonuclease FokI as a test system. To cleave DNA efficiently, FokI bridges two copies of an asymmetric sequence, invariably aligning the sites in parallel. On account of the fixed alignment, the topology of the DNA loop is set by the orientation of the sites along the DNA. We show that both the separation of the FokI sites and their orientation, altering, respectively, the twisting and the bending of the DNA needed to juxtapose the sites, have profound effects on the dynamics of the looping interaction. Surprisingly, the presence of a nick within the loop does not affect the observed rigidity of the DNA. In contrast, the introduction of a 4-nt gap fully relaxes all of the torque present in the system but does not necessarily enhance loop stability. FokI therefore employs torque to stabilise its DNA-looping interaction by acting as a ‘torsional’ catch bond

Type III restriction endonucleases are heterotrimeric:comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism

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

Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer

The FokI restriction endonuclease is a monomeric protein that recognizes an asymmetric sequence and cleaves both DNA strands at fixed loci downstream of the site. Its single active site is positioned initially near the recognition sequence, distant from its downstream target 13 nucleotides away. Moreover, to cut both strands, it has to recruit a second monomer to give an assembly with two active sites. Here, the individual steps in the FokI reaction pathway were examined by fluorescence resonance energy transfer (FRET). To monitor DNA binding and domain motion, a fluorescence donor was attached to the DNA, either downstream or upstream of the recognition site, and an acceptor placed on the catalytic domain of the protein. A FokI variant incapable of dimerization was also employed, to disentangle the signal due to domain motion from that due to protein association. Dimerization was monitored separately by using two samples of FokI labelled with donor and acceptor, respectively. The stopped-flow studies revealed a complete reaction pathway for FokI, both the sequence of events and the kinetics of each individual step

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission

Protein dynamics of the restriction endonuclease Fokl

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TstI, a Type II restriction-modification protein with DNA recognition, cleavage and methylation functions in a single polypeptide

Type II restriction–modification systems cleave and methylate DNA at specific sequences. However, the Type IIB systems look more like Type I than conven-tional Type II schemes as they employ the same pro-tein for both restriction and modification and for DNA recognition. Several Type IIB proteins, including the archetype BcgI, are assemblies of two polypeptides: one with endonuclease and methyltransferase roles, another for DNA recognition. Conversely, some IIB proteins express all three functions from separate segments of a single polypeptide. This study anal-ysed one such single-chain protein, TstI. Compar-ison with BcgI showed that the one- and the two-polypeptide systems differ markedly. Unlike the het-erologous assembly of BcgI, TstI forms a homote-tramer. The tetramer bridges two recognition sites before eventually cutting the DNA in both strands on both sides of the sites, but at each site the first double-strand break is made long before the second. In contrast, BcgI cuts all eight target bonds at two sites in a single step. TstI also differs from BcgI in either methylating or cleaving unmodified sites at similar rates. The site may thus be modified before TstI can make the second double-strand break. TstI MTase acts best at hemi-methylated sites

TstI, a Type II restriction-modification protein with DNA recognition, cleavage and methylation functions in a single polypeptide

Type II restriction–modification systems cleave and methylate DNA at specific sequences. However, the Type IIB systems look more like Type I than conven-tional Type II schemes as they employ the same pro-tein for both restriction and modification and for DNA recognition. Several Type IIB proteins, including the archetype BcgI, are assemblies of two polypeptides: one with endonuclease and methyltransferase roles, another for DNA recognition. Conversely, some IIB proteins express all three functions from separate segments of a single polypeptide. This study anal-ysed one such single-chain protein, TstI. Compar-ison with BcgI showed that the one- and the two-polypeptide systems differ markedly. Unlike the het-erologous assembly of BcgI, TstI forms a homote-tramer. The tetramer bridges two recognition sites before eventually cutting the DNA in both strands on both sides of the sites, but at each site the first double-strand break is made long before the second. In contrast, BcgI cuts all eight target bonds at two sites in a single step. TstI also differs from BcgI in either methylating or cleaving unmodified sites at similar rates. The site may thus be modified before TstI can make the second double-strand break. TstI MTase acts best at hemi-methylated sites

Multipartite control of the DNA translocase, Mfd

ATP-dependent nucleic acid helicases and translocases play essential roles in many aspects of DNA and RNA biology. In order to ensure that these proteins act only in specific contexts, their activity is often regulated by intramolecular contacts and interaction with partner proteins. We have studied the bacterial Mfd protein, which is an ATP-dependent DNA translocase that relocates or displaces transcription ECs in a variety of cellular contexts. When bound to RNAP, Mfd exhibits robust ATPase and DNA translocase activities, but when released from its substrate these activities are repressed by autoinhibitory interdomain contacts. In this work, we have identified an interface within the Mfd protein that is important for regulating the activity of the protein, and whose disruption permits Mfd to act indiscriminately at transcription complexes that lack the usual determinants of Mfd specificity. Our results indicate that regulation of Mfd occurs through multiple nodes, and that activation of Mfd may be a multi-stage process

Phagocytosed Polyhedrin-Cytokine Cocrystal Nanoparticles Provide Sustained Secretion of Bioactive Cytokines from Macrophages

Many cells possess the ability to engulf and incorporate particles by phagocytosis. This active process is characteristic of microorganisms as well as higher order species. In mammals, monocytes, macrophages, and microglia are among the so-called professional phagocytes. In addition, cells such as fibroblast and chondrocytes are classified as nonprofessional phagocytes. Professional phagocytes play important roles in both the innate and adaptive immune responses, wound healing, and tissue homeostasis. Consequently, these cells are increasingly studied as targets and vectors of therapeutic intervention to treat a range of diseases. Professional phagocytes are notoriously difficult to transfect limiting their study and manipulation. Consequently, efforts have shifted towards the development of nanoparticles to deliver a cargo to phagocytic cells via phagocytosis. However, this approach carries significant technical challenges, particularly for protein cargos. We have focused on the development of nanoscale cocrystalline protein depots, known as PODS®, that contain protein cargos, including cytokines. Here, we show that PODS are readily phagocytosed by nonprofessional as well as professional phagocytic cells and have attributes, such as highly sustained release of cargo, that suggest potential utility for the study and exploitation of phagocytic cells for drug delivery. Monocytes and macrophages that ingest PODS retain normal characteristics including a robust chemotactic response. Moreover, the PODS-cytokine cargo is secreted by the loaded cell at a level sufficient to modulate the behavior of surrounding nonphagocytic cells. The results presented here demonstrate the potential of PODS nanoparticles as a novel molecular tool for the study and manipulation of phagocytic cells and for the development of Trojan horse immunotherapy strategies to treat cancer and other diseases