The structure of the acto-myosin subfragment 1 complex: Results of searches using data from electron microscopy and x-ray crystallography

Surmises of how myosin subfragment 1 (S1) interacts with actin filaments in muscle contraction rest upon knowing the relative arrangement of the two proteins. Although there exist crystallographic structures for both S1 and actin, as well as electron microscopy data for the acto–S1 complex (AS1), modeling of this arrangement has so far only been done “by eye.” Here we report fitted AS1 structures obtained using a quantitative method that is both more objective and makes more complete use of the data. Using undistorted crystallographic results, the best-fit AS1 structure shows significant differences from that obtained by visual fitting. The best fit is produced using the F-actin model of Holmes et al. [Holmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature (London) 347, 44–49]. S1 residues at the AS1 interface are now found at a higher radius as well as being translated axially and rotated azimuthally. Fits using S1 plus loops missing from the crystal structure were achieved using a homology search method to predict loop structures. These improved fits favor an arrangement in which the loop at the 50- to 20-kDa domain junction of S1 is located near the N terminus of actin. Rigid-body movements of the lower 50-kDa domain, which further improve the fit, produce closure of the large 50-kDa domain cleft and bring conserved residues in the lower 50-kDa domain into an apparently appropriate orientation for close interaction with actin. This finding supports the idea that binding of ATP to AS1 at the end of the ATPase cycle disrupts the actin binding site by changing the conformation of the 50-kDa cleft of S1.

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments

The observation of light metal ions in nucleic acids crystals is generally a fortuitous event. Sodium ions in particular are notoriously difficult to detect because their X-ray scattering contributions are virtually identical to those of water and Na+…O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. We demonstrate here that replacement of Na+ by K+, Rb+ or Cs+ and precise measurements of anomalous differences in intensities provide a particularly sensitive method for detecting alkali metal ion-binding sites in nucleic acid crystals. Not only can alkali metal ions be readily located in such structures, but the presence of Rb+ or Cs+ also allows structure determination by the single wavelength anomalous diffraction technique. Besides allowing identification of high occupancy binding sites, the combination of high resolution and anomalous diffraction data established here can also pinpoint binding sites that feature only partial occupancy. Conversely, high resolution of the data alone does not necessarily allow differentiation between water and partially ordered metal ions, as demonstrated with the crystal structure of a DNA duplex determined to a resolution of 0.6 Å.

Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks, alkylations, or x-ray damage.

The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plus-Giemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions.