Vincent W. Coljee

DNA unzipped under a constant force exhibits multiple metastable intermediates.

Single molecule studies, at constant force, of the separation of double-stranded DNA into two separated single strands may provide information relevant to the dynamics of DNA replication. At constant applied force, theory predicts that the unzipped length as a function of time is characterized by jumps during which the strands separate rapidly, followed by long pauses where the number of separated base pairs remains constant. Here, we report previously uncharacterized observations of this striking behavior carried out on a number of identical single molecules simultaneously. When several single λ phage molecules are subject to the same applied force, the pause positions are reproducible in each. This reproducibility shows that the positions and durations of the pauses in unzipping provide a sequence-dependent molecular fingerprint. For small forces, the DNA remains in a partially unzipped state for at least several hours. For larger forces, the separation is still characterized by jumps and pauses, but the double-stranded DNA will completely unzip in less than 30 min.

Single Molecule Detection of Direct, Homologous, DNA/DNA Pairing

Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the “default” option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.

The structure of DNA overstretched from the 5′5′ ends differs from the structure of DNA overstretched from the 3′3′ ends

It has been suggested that the structure that results when double-stranded DNA (dsDNA) is pulled from the 3′3′ ends differs from that which results when it is pulled from the 5′5′ ends. In this work, we demonstrate, using λ phage dsDNA, that the overstretched states do indeed show different properties, suggesting that they correspond to different structures. For 3′3′ pulling versus 5′5′ pulling, the following differences are observed: (i) the forces at which half of the molecules in the ensemble have made a complete force-induced transition to single stranded DNA are 141 ± 3 pN and 122 ± 4 pN, respectively; (ii) the extension vs. force curve for overstretched DNA has a marked change in slope at 127 ± 3 pN for 3′3′ and 110 ± 3 pN for 5′5′; (iii) the hysteresis (H) in the extension vs. force curves at 150 mM NaCl is 0.3 ± 0.8 pN μm for 3′3′ versus 13 ± 8 pN for 5′5′; and (iv) 3′3′ and 5′5′ molecules show different changes in hysteresis due to interactions with β-cyclodextrin, a molecule that is known to form stable host-guest complexes with rotated base pairs, and glyoxal that is known to bind stably to unpaired bases. These differences and additional findings are well-accommodated by the corresponding structures predicted on theoretical grounds.

Reduced Susceptibility of Recombinant Polyclonal Antibodies to Inhibitory Anti-Variable Domain Antibody Responses

The immunogenicity of therapeutic Abs is a concern as anti-drug Abs may impact negatively on the pharmacodynamics and safety profile of Ab drugs. The factors governing induction of anti-drug Abs are not fully understood. In this study, we describe a model based on mouse-human chimeric Abs for the study of Ab immunogenicity in vivo. Six chimeric Abs containing human V regions and mouse C regions were generated from six human anti-Rhesus D Abs and the Ag-binding characteristics of the parental human Abs were retained. Analysis of the immune response toward the individual chimeric Abs revealed the induction of anti-variable domain Abs including anti-idiotypic Abs against some of these, thereby demonstrating the applicability of the model for studying anti-drug Ab responses in vivo. Immunization of BALB/c, C57, and outbred NMRI mice with a polyclonal composition consisting of all six chimeric Abs demonstrated that the immunogenicity of the individual Abs was haplotype dependent. Chimeric Abs, which were nonimmunogenic when administered individually, did not become immunogenic as part of the polyclonal composition, implying the absence of epitope spreading. Ex vivo Ab-binding studies established a clear correlation between the level of immunogenicity of the Abs comprised in the composition and the impact on the pharmacology of the Abs. These analyses demonstrate that under these conditions this polyclonal Ab composition was generally less susceptible to blocking Abs than the respective mAbs.

Back to the future: recombinant polyclonal antibody therapeutics.

Antibody therapeutics are one of the fastest growing classes of pharmaceuticals, with an annual US market over

Measurement of the salt-dependent stabilization of partially open DNA by Escherichia coli SSB protein

20 billion, developed to treat a variety of diseases including cancer, auto-immune and infectious diseases. Most are currently administered as a single molecule to treat a single disease; however there is mounting evidence that cocktails of multiple antibodies, each with a unique binding specificity and protective mechanism, may improve clinical efficacy. Here, we review progress in the development of oligoclonal combinations of antibodies to treat disease, focusing on identification of synergistic antibodies. We then discuss the application of modern antibody engineering technologies to produce highly potent antibody preparations, including oligoclonal antibody cocktails and truly recombinant polyclonal antibodies. Specific examples illustrating the synergy conferred by multiple antibodies will be provided for diseases caused by botulinum toxin, cancer and immune thrombocytopenia. The bioprocessing and regulatory options for these preparations will be discussed.

Tension on dsDNA bound to ssDNA-RecA filaments may play an important role in driving efficient and accurate homology recognition and strand exchange

The rezipping force of two complementary DNA strands under tension has been measured in the presence of Escherichia coli single-stranded-binding proteins under salt conditions ranging from 10– to 400 mM NaCl. The effectiveness of the binding protein in preventing rezipping is strongly dependent on salt concentration and compared with the salt dependence in the absence of the protein. At concentrations less than 50 mM NaCl, the protein prevents complete rezipping of λ-phage on the 2-s timescale of the experiment, when the ssDNA is under tensions as low as 3.5 ± 1 pN. For salt concentrations greater than 200 mM NaCl, the protein inhibits rezipping but cannot block rezipping when the tension is reduced below 6 ± 1.8 pN. This change in effectiveness as a function of salt concentration may correspond to salt-dependent changes in binding modes that were previously observed in bulk assays.

Complementary strand relocation may play vital roles in RecA-based homology recognition.

It is well known that during homology recognition and strand exchange the double stranded DNA (dsDNA) in DNA/RecA filaments is highly extended, but the functional role of the extension has been unclear. We present an analytical model that calculates the distribution of tension in the extended dsDNA during strand exchange. The model suggests that the binding of additional dsDNA base pairs to the DNA/RecA filament alters the tension in dsDNA that was already bound to the filament, resulting in a non-linear increase in the mechanical energy as a function of the number of bound base pairs. This collective mechanical response may promote homology stringency and underlie unexplained experimental results.

Changes in the tension in dsDNA alter the conformation of RecA bound to dsDNA–RecA filaments

RecA-family proteins mediate homologous recombination and recombinational DNA repair through homology search and strand exchange. Initially, the protein forms a filament with the incoming single-stranded DNA (ssDNA) bound in site I. The RecA–ssDNA filament then binds double-stranded DNA (dsDNA) in site II. Non-homologous dsDNA rapidly unbinds, whereas homologous dsDNA undergoes strand exchange yielding heteroduplex dsDNA in site I and the leftover outgoing strand in site II. We show that applying force to the ends of the complementary strand significantly retards strand exchange, whereas applying the same force to the outgoing strand does not. We also show that crystallographically determined binding site locations require an intermediate structure in addition to the initial and final structures. Furthermore, we demonstrate that the characteristic dsDNA extension rates due to strand exchange and free RecA binding are the same, suggesting that relocation of the complementary strand from its position in the intermediate structure to its position in the final structure limits both rates. Finally, we propose that homology recognition is governed by transitions to and from the intermediate structure, where the transitions depend on differential extension in the dsDNA. This differential extension drives strand exchange forward for homologs and increases the free energy penalty for strand exchange of non-homologs.

Probing the mechanical stability of DNA in the presence of monovalent cations.

The RecA protein is an ATPase that mediates recombination via strand exchange. In strand exchange a single-stranded DNA (ssDNA) bound to RecA binding site I in a RecA/ssDNA filament pairs with one strand of a double-stranded DNA (dsDNA) and forms heteroduplex dsDNA in site I if homology is encountered. Long sequences are exchanged in a dynamic process in which initially unbound dsDNA binds to the leading end of a RecA/ssDNA filament, while heteroduplex dsDNA unbinds from the lagging end via ATP hydrolysis. ATP hydrolysis is required to convert the active RecA conformation, which cannot unbind, to the inactive conformation, which can unbind. If dsDNA extension due to RecA binding increases the dsDNA tension, then RecA unbinding must decrease tension. We show that in the presence of ATP hydrolysis decreases in tension induce decreases in length whereas in the absence of hydrolysis, changes in tension have no systematic effect. These results suggest that decreases in force enhance dissociation by promoting transitions from the active to the inactive RecA conformation. In contrast, increases in tension reduce dissociation. Thus, the changes in tension inherent to strand exchange may couple with ATP hydrolysis to increase the directionality and stringency of strand exchange.