On the Mechanism of Homology Search by RecA Protein Filaments.

Genetic stability is a key factor in maintaining, survival, and reproduction of biological cells. It relies on many processes, but one of the most important is a homologous recombination, in which the repair of breaks in double-stranded DNA molecules is taking place with a help of several specific proteins. In bacteria, this task is accomplished by RecA proteins that are active as nucleoprotein filaments formed on single-stranded segments of DNA. A critical step in the homologous recombination is a search for a corresponding homologous region on DNA, which is called a homology search. Recent single-molecule experiments clarified some aspects of this process, but its molecular mechanisms remain not well understood. We developed a quantitative theoretical approach to analyze the homology search. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the homology search is presented. It is found that the search dynamics depends on the degree of extension of DNA molecules and on the size of RecA nucleoprotein filaments, in agreement with experimental single-molecule measurements of DNA pairing by RecA proteins. Our theoretical calculations, supported by extensive Monte Carlo computer simulations, provide a molecular description of the mechanisms of the homology search.

Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery.

The tuberculosis (TB) drug discovery pipeline is fueled by compounds identified in whole-cell screens against the causative agent, Mycobacterium tuberculosis Phenotypic screening enables the selection of molecules that inhibit essential cellular functions in live, intact bacilli grown under a chosen in vitro condition. However, deducing the mechanism of action (MOA), which is important to avoid promiscuous targets, often requires significant biological resources in a lengthy process that risks decoupling medicinal chemistry and biology efforts. Therefore, there is a need to develop methods enabling rapid MOA assessment of putative "actives" for triage decisions. Here, we describe a modified version of a bioluminescence reporter assay that allows nondestructive detection of compounds targeting either of two macromolecular processes in M. tuberculosis: cell wall biosynthesis or maintenance of DNA integrity. Coupling the luxCDABE operon from Photorhabdus luminescens to mycobacterial promoters driving expression of the iniBAC operon (PiniB-LUX) or the DNA damage-inducible genes, recA (PrecA-LUX) or radA (PradA-LUX), provided quantitative detection in real time of compounds triggering expression of any of these promoters over an extended 10- to 12-day incubation. Testing against known anti-TB agents confirmed the specificity of each reporter in registering the MOA of the applied antibiotic in M. tuberculosis, independent of bactericidal or bacteriostatic activity. Moreover, profiles obtained for experimental compounds indicated the potential to infer complex MOAs in which multiple cellular processes are disrupted. These results demonstrate the utility of the reporters for early triage of compounds based on the provisional MOA and suggest their application to investigate polypharmacology in known and experimental anti-TB agents.

A comparative study of multivariate and univariate hidden Markov modelings in time-binned single-molecule FRET data analysis.

We compare two different types of hidden Markov modeling (HMM) algorithms, e.g., multivariate HMM (MHMM) and univariate HMM (UHMM), for the analysis of time-binned single-molecule fluorescence energy transfer (smFRET) data. In MHMM, the original two channel signals, i.e., the donor fluorescence intensity (I(D)) and acceptor fluorescence intensity (I(A)), are simultaneously analyzed. However, in UHMM, only the calculated FRET trajectory is analyzed. On the basis of the analysis of both synthetic and experimental data, we find that, if the noise in the signal is described with a proper probability distribution, MHMM generally outperforms UHMM. We also show that, in the case of multiple trajectories, analyzing them simultaneously gives better results than averaging over individual analysis results.

Brownian ratchets driven by asymmetric nucleation of hydrolysis waves.

We propose a stochastic process wherein molecular transport is mediated by asymmetric nucleation of domains on a one-dimensional substrate, in contrast with molecular motors that hydrolyze nucleotide triphosphates and undergo conformational change. We show that asymmetric nucleation of hydrolysis waves on a track can also result in directed motion of an attached particle. Asymmetrically cooperative kinetics between hydrolyzed and unhydrolyzed states on each lattice site generate moving domain walls that push a particle sitting on the track. We use a novel fluctuating-frame, finite-segment mean field theory to accurately compute steady-state velocities of the driven particle and to discover parameter regimes yielding maximal domain wall flux, leading to optimal particle drift.

RecA mediated initial alignment of homologous DNA molecules displays apparent first order kinetics with little effect of heterology.

The mechanism and determinants of RecA mediated initial alignment of homologous DNA molecules were studied by performing Monte Carlo simulations of the dynamics of DNA molecules. The simulation procedure was used to assess the effect of heterologous DNA and dilution on the rate of formation and yield of homologous alignments. The results show that the apparent first order kinetic behavior and the impact of heterologous DNA, reported in literature [J. Biol. Chem. 261 (1986) 1025], can be observed even if the conversion of the initially aligned molecules into a stable joint is not rate-determining. The present study is the first step towards developing rigorous computational models to describe the process of homologous recombination, and theoretical frameworks to retrieve biophysical parameters of strand pairing and exchange proteins from in vitro assays of joint molecule formation.

DNA repair: getting past a lesion - at a cost.

Bacteria survive many types of synthesis-blocking DNA lesion by inducing a number of proteins that enable their polymerases to synthesize past a lesion, albeit at the cost of an increased mutation rate. This process has now been convincingly achieved in vitro, opening the way to a fuller understanding of the mechanism.

RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations.

Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.