Dynamics of DNA nicking and unwinding by the RepC-PcrA complex.

The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.

Jörg Langowski: his scientific legacy and the future it promises.

BACKGROUND: With the passing of Jörg Langowski 6 May 2017 in a sailplane accident, the scientific community was deprived of a strident and effective voice for DNA and chromatin molecular and computational biophysics, for open access publishing and for the creation of effective scientific research networks. METHODS: Here, after reviewing some of Jörg's key research contributions and ideas, we offer through the personal remembrance of his closest collaborators, a deep analysis of the major results of his research and the future directions they have engendered. CONCLUSIONS: The legacy of Jörg Langowski has been to propel a way of viewing biological function that considers living systems as dynamic and in three dimensions. This physical view of biology that he pioneered is now, finally, becoming established also because of his great effort.

Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding.

Replicative hexameric helicases are thought to unwind duplex DNA by steric exclusion (SE) where one DNA strand is encircled by the hexamer and the other is excluded from the central channel. However, interactions with the excluded strand on the exterior surface of hexameric helicases have also been shown to be important for DNA unwinding, giving rise to the steric exclusion and wrapping (SEW) model. For example, the archaeal Sulfolobus solfataricus minichromosome maintenance (SsoMCM) helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency. Using single-molecule FRET, we now show that the analogous Escherichia coli (Ec) DnaB helicase also interacts specifically with the excluded DNA strand during unwinding. Mutation of several conserved and positively charged residues on the exterior surface of EcDnaB resulted in increased interaction dynamics and states compared with wild type. Surprisingly, these mutations also increased the DNA unwinding rate, suggesting that electrostatic contacts with the excluded strand act as a regulator for unwinding activity. In support of this, experiments neutralizing the charge of the excluded strand with a morpholino substrate instead of DNA also dramatically increased the unwinding rate. Of note, although the stability of the excluded strand was nearly identical for EcDnaB and SsoMCM, these enzymes are from different superfamilies and unwind DNA with opposite polarities. These results support the SEW model of unwinding for EcDnaB that expands on the existing SE model of hexameric helicase unwinding to include contributions from the excluded strand to regulate the DNA unwinding rate.

Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules.

Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5'-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate.

Mechanism of allosteric inhibition of HIV-1 reverse transcriptase revealed by single-molecule and ensemble fluorescence.

Non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are routinely used to treat HIV-1 infection, yet their mechanism of action remains unclear despite intensive investigation. In this study, we developed complementary single-molecule fluorescence and ensemble fluorescence anisotropy approaches to discover how NNRTIs modulate the intra-molecular conformational changes and inter-molecular dynamics of RT-template/primer (T/P) and RT-T/P-dNTP complexes. We found that NNRTI binding to RT induces opening of the fingers and thumb subdomains, which increases the dynamic sliding motion of the enzyme on the T/P and reduces dNTP binding affinity. Further, efavirenz promotes formation of the E138-K101 salt bridge between the p51 and p66 subunits of RT, which contributes to opening of the thumb/fingers subdomains. Engineering a more polar salt bridge between p51 and p66 resulted in even greater increases in the thumb/fingers opening, RT sliding, dNTP binding disruption and in vitro and in vivo RT inhibition than were observed with wild-type RT. We also observed that K103N, a clinically relevant NNRTI resistance mutation, does not prevent binding between efavirenz and RT-T/P but instead allows formation of a stable and productive RT-T/P-dNTP complex, possibly through disruption of the E138-K101 salt bridge. Collectively, these data describe unique structure-activity-resistance relationships that could be exploited for drug development.

Early integration of the individual student in academic activities: a novel classroom concept for graduate education in molecular biophysics and structural biology.

BACKGROUND: A key challenge in interdisciplinary research is choosing the best approach from a large number of techniques derived from different disciplines and their interfaces. RESULTS: To address this challenge in the area of Biophysics and Structural Biology, we have designed a graduate level course to teach students insightful use of experimental biophysical approaches in relationship to addressing biological questions related to biomolecular interactions and dynamics. A weekly seminar and data and literature club are used to compliment the training in class. The course contains wet-laboratory experimental demonstration and real-data analysis as well as lectures, grant proposal preparation and assessment, and student presentation components. Active student participation is mandatory in all aspects of the class. Students prepare materials for the class receiving individual and iterative feedback from course directors and local experts generating high quality classroom presentations. CONCLUSIONS: The ultimate goal of the course is to teach students the skills needed to weigh different experimental approaches against each other in addressing a specific biological question by thinking and executing academic tasks like faculty.

PcrA-mediated disruption of RecA nucleoprotein filaments--essential role of the ATPase activity of RecA.

The essential DNA helicase, PcrA, regulates recombination by displacing the recombinase RecA from the DNA. The nucleotide-bound state of RecA determines the stability of its nucleoprotein filaments. Using single-molecule fluorescence approaches, we demonstrate that RecA displacement by a translocating PcrA requires the ATPase activity of the recombinase. We also show that in a 'head-on collision' between a polymerizing RecA filament and a translocating PcrA, the RecA K72R ATPase mutant, but not wild-type RecA, arrests helicase translocation. Our findings demonstrate that translocation of PcrA is not sufficient to displace RecA from the DNA and assigns an essential role for the ATPase activity of RecA in helicase-mediated disruption of its filaments.

Monoubiquitinated histone H2A destabilizes photolesion-containing nucleosomes with concomitant release of UV-damaged DNA-binding protein E3 ligase.

How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4B(DDB2). We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4B(DDB2) E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4B(DDB2) from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.

Optimal practices for surface-tethered single molecule total internal reflection fluorescence resonance energy transfer analysis.

Single molecule fluorescence microscopy can be used to follow the mechanics of molecular biology processes in real time. However, many factors, from flow cell preparation to improper data analysis can negatively impact single molecule fluorescence resonance energy transfer (smFRET) experiments. Here, we describe some best practices for ensuring that smFRET data are of the highest quality. In addition to instrumentation, we describe sample preparation and data analysis.

Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding.

The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5'-tail is shown to provide stability to the MCM complex on DNA and contribute to unwinding. Mutations in a positively charged patch on the exterior surface of the MCM hexamer destabilize this interaction, alter the path of the displaced 5'-tail DNA and reduce unwinding. DNA footprinting and single-molecule fluorescence experiments support a previously unrecognized wrapping of the 5'-tail. This mode of hexameric helicase DNA unwinding is termed the steric exclusion and wrapping (SEW) model, where the 3'-tail is encircled by the helicase while the displaced 5'-tail wraps around defined paths on the exterior of the helicase. The novel wrapping mechanism stabilizes the MCM complex in a positive unwinding mode, protects the displaced single-stranded DNA tail and prevents reannealing.

Paracrine and epigenetic control of trophectoderm differentiation from human embryonic stem cells: the role of bone morphogenic protein 4 and histone deacetylases.

Our understanding of paracrine and epigenetic control of trophectoderm (TE) differentiation is limited by available models of preimplantation human development. Simple, defined media for selective TE differentiation of human embryonic stem cells (hESCs) were developed, enabling mechanistic studies of early placental development. Paracrine requirements of preimplantation human development were evaluated with hESCs by measuring lineage-specific transcription factor expression levels in single cells and morphological transformation in response to selected paracrine and epigenetic modulators. Bone morphogenic protein 4 (BMP4) addition to feeder-free pluripotent stem cells on matrigel frequently formed CDX2-positive TE. However, BMP4 or activin A inhibition alone also produced a mix of mesoderm and extraembryonic endoderm under these conditions. Further, BMP4 failed to form TE from adherent hESC maintained in standard feeder-dependent monolayers. Given that the efficiency and selectivity of BMP4-induced TE depended on medium components, we developed a basal medium containing insulin and heparin. In this medium, BMP4 induction of TE was dose dependent and with activin A inhibition by SB431542 (SB), approached 100% of cells. This paracrine stimulation of pluripotent cells transformed colony morphology from a cuboidal to squamous epithelium quantitatively on day 3, and produced significant multinucleated syncytiotrophoblasts by day 8. Addition of trichostatin A, a histone deacetylase (HDAC) inhibitor, reduced HDAC3, histone H3K9 methylation, and slowed differentiation in a dose-dependent manner. Modulators of BMP4- or HDAC-dependent signaling might adversely influence the timing and viability of early blastocyst developed in vitro. Since blastocyst development is synchronized to uterine receptivity, epigenetic regulators of TE differentiation might adversely affect implantation in vivo.

Dynamics of individual polymers using microfluidic based microcurvilinear flow.

Polymer dynamics play an important role in a diversity of fields including materials science, physics, biology and medicine. The spatiotemporal responses of individual molecules such as biopolymers have been critical to the development of new materials, the expanded understanding of cell structures including cytoskeletal dynamics, and DNA replication. The ability to probe single molecule dynamics however is often limited by the availability of small-scale technologies that can manipulate these systems to uncover highly intricate behaviors. Advances in micro- and nano-scale technologies have simultaneously provided us with valuable tools that can interface with these systems including methods such as microfluidics. Here, we report on the creation of micro-curvilinear flow through a small-scale fluidic approach, which we have been used to impose a flow-based high radial acceleration ( approximately 10(3) g) on individual flexible polymers. We were able to employ this microfluidic-based approach to adjust and control flow velocity and acceleration to observe real-time dynamics of fluorescently labeled lambda-phage DNA molecules in our device. This allowed us to impose mechanical stimulation including stretching and bending on single molecules in localized regimes through a simple and straightforward technology-based method. We found that the flexible DNA molecules exhibited multimodal responses including distinct conformations and controllable curvatures; these characteristics were directly related to both the elongation and bending dynamics dictated by their locations within the curvilinear flow. We analyzed the dynamics of these individual molecules to determine their elongation strain rates and curvatures ( approximately 0.09 microm(-1)) at different locations in this system to probe the individual polymer structural response. These results demonstrate our ability to create high radial acceleration flow and observe real-time dynamic responses applied directly to individual DNA molecules. This approach may also be useful for studying other biologically based polymers including additional nucleic acids, actin filaments, and microtubules and provide a platform to understand the material properties of flexible polymers at a small scale.

Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.

Post-translational modifications (PTMs) of histones play a role in modifying chromatin structure for DNA-templated processes in the eukaryotic nucleus, such as transcription, replication, recombination and repair; thus, histone PTMs are considered major players in the epigenetic control of these processes. Linking specific histone PTMs to gene expression is an arduous task requiring large amounts of highly purified and natively modified histones to be analyzed by various techniques. We have developed robust and complementary procedures, which use strong protein denaturing conditions and yield highly purified core and linker histones from unsynchronized proliferating, M-phase arrested and butyrate-treated cells, fully preserving their native PTMs without using enzyme inhibitors. Cell hypotonic swelling and lysis, nuclei isolation/washing and chromatin solubilization under mild conditions are bypassed to avoid compromising the integrity of histone native PTMs. As controls for our procedures, we tested the most widely used conventional methodologies and demonstrated that they indeed lead to drastic histone dephosphorylation. Additionally, we have developed methods for preserving acid-labile histone modifications by performing non-acid extractions to obtain highly purified H3 and H4. Importantly, isolation of histones H3, H4 and H2A/H2B is achieved without the use of HPLC. Functional supercoiling assays reveal that both hyper- and hypo-phosphorylated histones can be efficiently assembled into polynucleosomes. Notably, the preservation of fully phosphorylated mitotic histones and their assembly into polynucleosomes should open new avenues to investigate an important but overlooked question: the impact of mitotic phosphorylation in chromatin structure and function.

Structure and dynamics of single DNA molecules manipulated by magnetic tweezers and or flow.

Here we describe the use of magnetic tweezers and or microfluidics to manipulate single DNA molecules. We describe experiment which employ magnetic tweezers coupled to an inverted microscope as well as the use of a magnetic tweezers setup with an upright microscope. Using a chamber prepared via soft lithography, we also describe a microfluidic device for the manipulation of individual DNA molecules. Finally, we present some past successful examples of using these approaches to elucidate unique information about protein-nucleic acid interactions.

Expedient placement of two fluorescent dyes for investigating dynamic DNA protein interactions in real time.

Many questions in molecular and cellular biology can be reduced to questions about 'who talks to whom, when and how frequently'. Here, we review approaches we have used with single-pair fluorescence resonance energy transfer (spFRET) to follow the motions between two well-placed fluorescent probes to ask similar questions. We describe two systems. We have used a nucleosomal system in which the naked DNA molecule has the acceptor and donor dyes too far apart for FRET to occur whereas the dyes are close enough in the reconstituted nucleosome for FRET. As these individual nucleosomes were tethered on a surface, we could follow dynamics in the repositioning of these two dyes, inferring that nucleosomes stochastically and reversibly open and close. These results imply that most of the DNA on the nucleosome can be sporadically accessible to regulatory proteins and proteins that track the DNA double helix. In the case of following the binding of recombination protein RecA to double-stranded DNA (dsDNA) and the RecA filament displacement by DNA helicase motor PcrA, the dsDNA template is prepared with the two dyes close enough to each other to generate high FRET. Binding of the RecA molecules to form a filament lengthens the dsDNA molecule 1.5-fold and reduces the FRET accordingly. Once added, DNA motor protein helicase PcrA can displace the RecA filament with concomitant return of the DNA molecule to its original B-form and high FRET state. Thus, appropriately placed fluorescent dyes can be used to monitor conformational changes occurring in DNA and or proteins and provide increased sensitivity for investigating dynamic DNA-protein interactions in real time.

cAMP signaling induces rapid loss of histone H3 phosphorylation in mammary adenocarcinoma-derived cell lines.

The phosphorylation of histone H3 is known to play a role in regulation of transcription as well as preparation of chromosomes for mitosis. Various signaling cascades induce H3 phosphorylation, particularly at genes activated by these pathways. In this study, we show that signaling can also have the opposite effect. Activators of cAMP signaling induce a rapid and potent loss of H3 phosphorylation. This effect is not mediated through a cAMP metabolite since a membrane-permeable form of AMP had no effect on H3 phosphorylation and a phosphodiesterase-resistant cAMP analog efficiently reduced it. cAMP is also the likely regulator of H3 phosphorylation under physiological conditions since only supra-pharmacological doses of cGMP induce the loss of H3 phosphorylation. The loss of phosphorylation is specific for histone H3 since we do not observe drastic losses in total phosphorylation of other histones. In addition, other H3 modifications are unaffected with the exception of lysine 9 methylation, which is elevated. Analysis of cell growth and cell cycle shows that cAMP signaling inhibits cell growth and arrests cells at both G1 and G2/M. Similar effects of cAMP signaling on H3 phosphorylation are observed in a variety of mammary adenocarcinoma-derived cell lines. In syngeneic human breast-derived cell lines, one diploid and non-transformed, the other derived from a ductal carcinoma, the loss of H3 phosphorylation is significantly more sensitive to cAMP concentration in the transformed cell line.

DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange.

PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions.

Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

Single-molecule approaches reveal the idiosyncrasies of RNA polymerases.

Recently developed single-molecule techniques have provided new insights into the function of one of the most complex and highly regulated processes in the cell--the transcription of the DNA template into RNA. This review discusses methods and results from this emerging field, and it puts them in perspective of existing biochemical and structural data.

Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by single-pair fluorescence resonance energy transfer.

The nucleosome core particle, the basic repeated structure in chromatin fibers, consists of an octamer of eight core histone molecules, organized as dimers (H2A/H2B) and tetramers [(H3/H4)2] around which DNA wraps tightly in almost two left-handed turns. The nucleosome has to undergo certain conformational changes to allow processes that need access to the DNA template to occur. By single-pair fluorescence resonance energy transfer, we demonstrate fast, long-range, reversible conformational fluctuations in nucleosomes between two states: fully folded (closed), with the DNA wrapped around the histone core, or open, with the DNA significantly unraveled from the histone octamer. The brief excursions into an extended open state may create windows of opportunity for protein factors involved in DNA transactions to bind to or translocate along the DNA.