Robert A. Forties

Histone fold modifications control nucleosome unwrapping and disassembly

Nucleosomes are stable DNA–histone protein complexes that must be unwrapped and disassembled for genome expression, replication, and repair. Histone posttranslational modifications (PTMs) are major regulatory factors of these nucleosome structural changes, but the molecular mechanisms associated with PTM function remains poorly understood. Here we demonstrate that histone PTMs within distinct structured regions of the nucleosome directly regulate the inherent dynamic properties of the nucleosome. Precise PTMs were introduced into nucleosomes by chemical ligation. Single molecule magnetic tweezers measurements determined that only PTMs near the nucleosome dyad increase the rate of histone release in unwrapped nucleosomes. In contrast, FRET and restriction enzyme analysis reveal that only PTMs throughout the DNA entry–exit region increase unwrapping and enhance transcription factor binding to nucleosomal DNA. These results demonstrate that PTMs in separate structural regions of the nucleosome control distinct dynamic events, where the dyad regulates disassembly while the DNA entry–exit region regulates unwrapping. These studies are consistent with the conclusion that histone PTMs may independently influence nucleosome dynamics and associated chromatin functions.

Human MSH2 (hMSH2) Protein Controls ATP Processing by hMSH2-hMSH6

Background: The hMSH2-hMSH6 heterodimer must coordinate mismatch binding with dual site adenosine nucleotide processing. Results: An hMSH2-magnesium-ADP complex inhibits ATP hydrolysis by both the hMSH2 and hMSH6 subunits. Conclusion: hMSH2 regulates adenosine nucleotide processing by the hMSH2-hMSH6 mismatch recognition heterodimer. Significance: Understanding the molecular mechanism of hMSH2-hMSH6 function is crucial for elucidating the role of the mismatch repair pathway in human tumorigenesis. The mechanics of hMSH2-hMSH6 ATP binding and hydrolysis are critical to several proposed mechanisms for mismatch repair (MMR), which in turn rely on the detailed coordination of ATP processing between the individual hMSH2 and hMSH6 subunits. Here we show that hMSH2-hMSH6 is strictly controlled by hMSH2 and magnesium in a complex with ADP (hMSH2(magnesium-ADP)-hMSH6). Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which then enhances ATP binding by hMSH2. Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for ∼8 min. These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer.

The flexibility of locally melted DNA

Protein-bound duplex DNA is often bent or kinked. Yet, quantification of intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA cyclization experiments have indicated that DNA may form sharp bends more easily than predicted by the established worm-like chain (WLC) model. One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges. We have expanded this model to incorporate sequence and temperature dependence of the local melting, and tested it for three sequences at temperatures from 23°C to 42°C. We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures. However, these bubbles are not flexible enough to explain the recently observed very sharp bends in DNA.

RAD51 protein ATP cap regulates nucleoprotein filament stability.

Background: RAD51 and RecA homologs form a nucleoprotein filament (NPF) that includes an ATP cap, which sandwiches the adenosine nucleotide. Results: The HsRAD51(D316K) ATP cap substitution dramatically enhances recombinase and NPF stability. Conclusion: The HsRAD51(Asp-316) residue forms a salt bridge with ATP that allows more rapid protein turnover. Significance: HsRAD51(D316K) provides a useful reagent for the study of recombinase function in physiological conditions. RAD51 mediates homologous recombination by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the ATP γ-phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position. We demonstrate that substitution of human RAD51(Asp-316) with lysine (HsRAD51(D316K)) decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) (MvRAD51(D302K)) and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(Asp-316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity.

In situ characterization of high-intensity laser beams on OMEGA

This article details a means of inferring high-intensity laser beam shapes as applied to the 60-beam ultraviolet (UV) (351nm) OMEGA laser system [T. R. Boehly et al., Opt. Commun. 133, 495 (1997)]. Measurements of the shape, location, and relative fluence of beams on the OMEGA laser system are obtained from x-ray images of the emission from 4‐mm‐diam, Au-coated pointing targets irradiated by the focused beams. The images are digitally recorded with an array of up to eight pinhole cameras. The image of each beam is corrected for the effect of view angle and conversion efficiency from UV to x rays, and fit to an elliptical super-Gaussian. The best-fit values from multiple views are combined to obtain values of the beam’s shape, beam-to-beam relative peak fluence, beam position, and errors thereof. This method allows the beam-to-beam balance and beam pointing to be further improved, if so desired, by providing measurements of these quantities at the target.

Modeling the interplay of single-stranded binding proteins and nucleic acid secondary structure

MOTIVATION There are many important proteins which bind single-stranded nucleic acids, such as the nucleocapsid protein in HIV and the RecA DNA repair protein in bacteria. The presence of such proteins can strongly alter the secondary structure of the nucleic acid molecules. Therefore, accurate modeling of the interaction between single-stranded nucleic acids and such proteins is essential to fully understand many biological processes. RESULTS We develop a model for predicting nucleic acid secondary structure in the presence of single-stranded binding proteins, and implement it as an extension of the Vienna RNA Package. All parameters needed to model nucleic acid secondary structures in the absence of proteins have been previously determined. This leaves the footprint and sequence-dependent binding affinity of the protein as adjustable parameters of our model. Using this model we are able to predict the probability of the protein binding at any position in the nucleic acid sequence, the impact of the protein on nucleic acid base pairing, the end-to-end distance distribution for the nucleic acid and FRET distributions for fluorophores attached to the nucleic acid. AVAILABILITY Source code for our modified version of the Vienna RNA package is freely available at http://bioserv.mps.ohio-state.edu/Vienna+P, implemented in C and running on Linux.

Anomalous scaling in nanopore translocation of structured heteropolymers

Translocation through nanopores has emerged as a new experimental technique to probe the physical properties of biomolecules. The question of how the typical translocation time for a single unstructured polymer depends on its length has already triggered many theoretical and computational studies. Here, we address the same question, but for structured RNA molecules where the breaking of base-pairing patterns is the main barrier for translocation. Within a simple model, we calculate the typical time for single-stranded RNAs with random sequences to translocate through an idealized nanopore. It is believed that with respect to secondary structure formation, such a random RNA is typically either frozen into a single dominant structure (glassy phase) or many different structures with similar total energies coexist (molten phase), depending on the temperature and the base-pairing energetics. We find that these two phases can be clearly distinguished by their translocation behavior in the absence of an external voltage bias. In both cases, the typical translocation time depends on the sequence length as a power law with an exponent that exceeds the naively expected exponent of two for purely diffusive translocation. However, whereas in the molten phase the exponent is constant, at a value of 5/2, the exponent increases rapidly with decreasing temperature in the glassy phase. We explain the behavior in the molten phase and the qualitative trend in the glassy phase theoretically.

Detection of Charged Particles with Charge Injection Devices

A method for using charge injection devices (CIDs) for detection of high-energy charged particles from inertial-confinement fusion reactions is described. Because of the relatively small depletion region of the CID camera (depletion depth of approximately 7 mum), aluminum foils are placed in front of the device to reduce the energy of the charged particles and maximize the energy deposited in the CID. Simultaneous measurements of (2)H(d,p)(3)H protons with a CID and a surface barrier detector indicate that the CID is an efficient detector of charged fusion products. Tests using high energy alpha particles emitted from a radium-226 source are also reported.

RNA Secondary Structure Prediction in the Presence of Single-Stranded Binding Proteins

Secondary structure prediction of RNA molecules is a problem in Bioinformatics with several well established solutions. However, while the approaches to RNA secondary structure prediction are successful in describing RNA molecules in vitro, RNA molecules in vivo often assume their secondary structure while interacting with many other consitutents of the cell. One of those constitutents that have the ability to significantly alter RNA secondary structure formation are proteins which bind single-stranded nucleic acids, such as the nucleocapsid protein in HIV or the RecA DNA repair protein in bacteria. We extend established secondary structure prediction methods to explicitly include the effect of such interactions. Using our model we are able to predict the probability of proteins binding at any position in the nucleic acid sequence as well as the impact of the protein on nucleic acid base pairing and on the end-to-end distance distribution of the nucleic acid.