Using specular reflection of neutrons, we investigate for the first time the structure of a single dimyristoylphosphatidylcholine bilayer adsorbed to a planar quartz surface in an aqueous environment. We demonstrate that the bilayer is strongly adsorbed to the quartz surface and is stable to phase state changes as well as exchange of the bulk aqueous phase. Our results show that the main phase transition is between the L alpha phase and the metastable L beta'* phase, with formation of the P beta' ripple phase prevented by lateral stress on the adsorbed bilayer. By performing contrast variation experiments, we are able to elucidate substantial detail in the interfacial structure. We measure a bilayer thickness of 43.0 +/- 1.5 A in the L alpha phase (T = 31 degrees C) and 46.0 +/- 1.5 A in the L beta'* phase (T = 20 degrees C). The polar head group is 8.0 +/- 1.5 A thick in the L alpha phase. The water layer between the quartz and bilayer is 30 +/- 10 A for the lipid in both the L alpha and L'* phase. Our results agree well with those previously reported from experiments using lipid vesicles and monolayers, thus establishing the feasibility of our experimental methods.

DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations.Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable Bacillus DNA polymerase I. The 6-bp extension involves a 20-Å translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal. In addition, we obtained the structure of a ''closed'' conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an ''open'' conformation. Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail. The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site. We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed.

Accurate DNA replication is essential for genomic stability. One mechanism by which high-fidelity DNA polymerases maintain replication accuracy involves stalling of the polymerase in response to covalent incorporation of mismatched base pairs, thereby favoring subsequent mismatch excision. Some polymerases retain a "short-term memory" of replication errors, responding to mismatches up to four base pairs in from the primer terminus. Here we a present a structural characterization of all 12 possible mismatches captured at the growing primer terminus in the active site of a polymerase. Our observations suggest four mechanisms that lead to mismatch-induced stalling of the polymerase. Furthermore, we have observed the effects of extending a mismatch up to six base pairs from the primer terminus and find that long-range distortions in the DNA transmit the presence of the mismatch back to the enzyme active site, suggesting the structural basis for the short-term memory of replication errors.