DNA polymerases copy DNA templates with remarkably high fidelity, checking for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps. Despite extensive biochemical, genetic and structural studies, the mechanism by which nucleotides are correctly incorporated is not known. Here we present high-resolution crystal structures of a thermostable bacterial (Bacillus stearothermophilus) DNA polymerase I large fragments with DNA primer templates bound productively at the polymerase active site. The active site retains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation. The polymerase also retains its ability to discriminate between correct and incorrectly paired nucleotides in the crystal. Comparison of the structures of successively translocated complexes allows the structural features for the sequence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions with the first four to five base pairs in the minor groove, location of the terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch from B-form to underwound A-form DNA at the polymerase active site.

The M 2 protein from inf luenza A virus forms proton-selective channels that are essential to viral function and are the target of the drug amantadine. Cys scanning was used to generate a series of mutants with successive substitutions in the transmembrane segment of the protein, and the mutants were expressed in Xenopus laevis oocytes. The effect of the mutations on reversal potential, ion currents, and amantadine resistance were measured. Fourier analysis revealed a periodicity consistent with a four-stranded coiled coil or helical bundle. A three-dimensional model of this structure suggests a possible mechanism for the proton selectivity of the M 2 channel of inf luenza virus.Ion channels are responsible for the rapid and efficient conduction of ions across phospholipid bilayers. They are generally highly selective for their permeant ions, and are gated by voltage or ligands (1). Although a number of high-resolution structures are available for hemolysins (2) and porins (3)-channel-like proteins that form large, nonselective poresstructural analysis of more selective ion channel proteins is at an early stage. Sequence analysis and low-resolution diffraction data indicate that their conduction pathways often consist of bundles of ␣-helices (4, 5), but the determination of high-resolution structures of channel proteins has been hampered by their limited availability and large size.M 2 from influenza virus is an essential component of the viral envelope and forms a highly selective, pH-regulated proton channel that is the target of the anti-influenza drug amantadine (6-9). The influenza virus enters cells through internalization into the endocytic pathway, with virus uncoating taking place in endosomal compartments. The M 2 ion channel activity permits protons to enter the virion interior, and this acidification weakens the interactions of the matrix protein (M 1 ) with the ribonucleoprotein core (10). By comparison to the channels of excitable tissues, M 2 is quite small (97 residues) and contains but one hydrophobic stretch of 18 residues believed to form a transmembrane (TM) helix (residues 26-43). A wealth of experimental evidence indicates that the M 2 channel DPL 26 VVAASIIGILHLILWIL 43 D consists of a tetrameric array of parallel, TM peptides with their N termini directed toward the outside of the virus (6-9). A synthetic 25-residue peptide spanning the hydrophobic region forms amantadine-sensitive proton channels, indicating that the determinants for assembly of the channel lie within this TM peptide (11). Further, CD spectroscopy indicates that this peptide forms ␣-helices in membranes (12). Thus, the TM region of the channel appears to consist of a parallel bundle of ␣-helices.Here we describe the use of Cys-scanning mutagenesis (13,17,18) to obtain more detailed information concerning the arrangement of the TM helices within the tetrameric pore. A similar method has been used previously to infer the probable structures of other homo-oligomeric TM proteins, including glycophorin (14) and phosph...

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This structure represents the highest resolution view of a Pol I enzyme obtained to date. Comparison of the three Pol I structures reveals no compelling evidence for many of the specific interactions that have been proposed to induce thermostability, but suggests that thermostability arises from innumerable small changes distributed throughout the protein structure. The polymerase domain is highly conserved in all three proteins. The N-terminal domains are highly divergent in sequence, but retain a common fold. When present, the 3'-5' proofreading exonuclease activity is associated with this domain. Its absence is associated with changes in catalytic residues that coordinate the divalent ions required for activity and in loops connecting homologous secondary structural elements. In BF, these changes result in a blockage of the DNA-binding cleft.

A polymorphic DNA marker has been found genetically linked, in a set of 39 human families, to an autosomal recessive gene that causes cystic fibrosis (CF), a disease affecting one in 2000 Caucasian children. The DNA marker (called DOCRI-917) is also linked to the PON locus, which by independent evidence is linked to the CF locus. The best estimates of the genetic distances are 5 centimorgans between the DNA marker and PON and 15 centimorgans between the DNA marker and the CF locus, meaning that the location of the disease gene has been narrowed to about 1 percent of the human genome (about 30 million base pairs). Although the data are consistent with the interpretation that a single locus causes cystic fibrosis, the possibility of genetic heterogeneity remains. The discovery of a linked DNA polymorphism is the first step in molecular analysis of the CF gene and its causative role in the disease. LAP-CHEE TsuI