Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion

E. coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair. Using small synthetic junctions, we show that junction recognition is structure-specific and occurs in the absence of metal cofactors. In the presence of Mg2+, Holliday junctions are resolved by the introduction of symmetrically related nicks at the 3' side of thymine residues. The nicked duplex products are repaired by the action of DNA ligase. Within the RuvC-Holliday junction complex, the DNA is distorted such that 2 of the 4 strands become hypersensitive to hydroxyl radical attack. The ionic requirements of binding, hydroxyl radical sensitivity, and strand cleavage indicate three distinct steps in the mechanism of RuvC-mediated Holliday junction resolution: structure-specific recognition, DNA distortion, and sequence-dependent cleavage.     

Structural alterations and conformational dynamics in Holliday junctions induced by binding of a site-specific recombinase

Binding of a cleavage-incompetent mutant of the Flp recombinase induces a roughly square-planar geometry in synthetic immobile Holliday junctions. The branch points, which are rigidly fixed in these junctions in their free forms, tend to be more flexible in their protein-bound forms. Our results (1) suggest a plausible mechanism for the switching of the recombination complex from the Holliday-forming mode to the Holliday-resolving mode, (2) provide a rationale for previous observations that Flp resolves preformed immobile Holliday structures in the parental or in the recombinant mode in a relatively unbiased manner, and (3) accommodate two modes of DNA cleavage by Flp (transhorizontal or transdiagonal) in Holliday substrates.     

Absolute quantitation of tissue phospholipids using 31P NMR spectroscopy

31P NMR spectroscopy has been used to quantitate phospholipids in tissue extracts without requiring their physical separation. Concentrated lipids were dissolved in chloroform-methanol-water 100:36:9 (v/v/v) containing a cesium salt of (ethylenedinitrilo)tetraacetic acid. Tri-n-butyl phosphate was added at the beginning of lipid extraction as an NMR internal standard, permitting the absolute quantitation of phospholipids in mumoles per gram of tissue. For efficient data collection, 10 mM chromium(III) acetylacetonate was included to promote relaxation. It was found that spectral peak separations could be optimized by manipulating the sample temperature. Phospholipid levels determined by NMR agreed with colorimetric measurements and literature values for rat liver and brain. Using a 0.5-1 g tissue sample and 800 averages (2 h acquisition), the coefficient of variation for total phospholipids was 2-3%.     

Sequence specificity and biochemical characterization of the RusA Holliday junction resolvase of Escherichia coli

The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates in recombination and DNA repair. Analysis of its subunit structure revealed that the native protein is a dimer. Its resolution activity was investigated using synthetic X-junctions with homologous cores. Resolution occurs by dual strand incision predominantly 5' of CC dinucleotides located symmetrically. A junction lacking homology is not resolved. The efficiency of resolution is related inversely to the number of base pairs in the homologous core, which suggests that branch migration is rate-limiting. Inhibition of resolution at high ratios of protein to DNA suggests that binding of RusA may immobilize the junction point at non-cleavable sites. Resolution is stimulated by alkaline pH and by Mn2+. The protein is unstable in the absence of substrate DNA and loses approximately 80% of its activity within 1 min under standard reaction conditions. DNA binding stabilizes the activity. Junction resolution is inhibited in the presence of RuvA. This observation probably explains why RusA is unable to promote efficient recombination and DNA repair in ruvA+ strains unless it is expressed at a high level.     

Practical model fitting approaches to the direct extraction of NMR parameters simultaneously from all dimensions of multidimensional NMR spectra

A maximum likelihood (ML)-based approach has been established for the direct extraction of NMR parameters (e.g., frequency, amplitude, phase, and decay rate) simultaneously from all dimensions of a D-dimensional NMR spectrum. The approach, referred to here as HTFD-ML (hybrid time frequency domain maximum likelihood), constructs a time-domain model composed of a sum of exponentially-decaying sinusoidal signals. The apodized Fourier transform of this time-domain signal is a model spectrum that represents the 'best-fit' to the equivalent frequency-domain data spectrum. The desired amplitude and frequency parameters can be extracted directly from the signal model constructed by the HTFD-ML algorithm. The HTFD-ML approach presented here, as embodied in the software package CHIFIT, is designed to meet the challenges posed by model fitting of D-dimensional NMR data sets, where each consists of many data points (10(8) is not uncommon) encoding information about numerous signals (up to 10(5) for a protein of moderate size) that exhibit spectral overlap. The suitability of the approach is demonstrated by its application to the concerted analysis of a series of ten 2D 1H-15N HSQC experiments measuring 15N T1 relaxation. In addition to demonstrating the practicality of performing maximum likelihood analysis on large, multidimensional NMR spectra, the results demonstrate that this parametric model-fitting approach provides more accurate amplitude and frequency estimates than those obtained from conventional peak-based analysis of the FT spectrum. The improved performance of the model fitting approach derives from its ability to take into account the simultaneous contributions of all signals in a crowded spectral region (deconvolution) as well as to incorporate prior knowledge in constructing models to fit the data.     

Polarization-enhanced NMR spectroscopy of biomolecules in frozen solution

Large dynamic nuclear polarization signal enhancements (up to a factor of 100) were obtained in the solid-state magic-angle spinning nuclear magnetic resonance (NMR) spectra of arginine and the protein T4 lysozyme in frozen glycerol-water solutions with the use of dynamic nuclear polarization. Polarization was transferred from the unpaired electrons of nitroxide free radicals to nuclear spins through microwave irradiation near the electron paramagnetic resonance frequency. This approach may be a generally applicable signal enhancement scheme for the high-resolution solid-state NMR spectroscopy of biomolecules.