contributed equally to this work DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3¢-tailed DNA duplex reveal a contact region that covers a signi®cant region of the substrate both in the presence and absence of a non-hydrolysable analogue of ATP, ADPNP. However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single-stranded DNA translocation activities intact. These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support aǹ active' mechanism for PcrA that involves two distinct ATP-dependent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.

Crystal structures and biochemical analyses of PcrA helicase provide evidence for a model for processive DNA unwinding that involves coupling of single-stranded DNA (ssDNA) tracking to a duplex destabilization activity. The DNA tracking model invokes ATP-dependent flipping of bases between several pockets on the enzyme formed by conserved aromatic amino acid residues. We have used site-directed mutagenesis to confirm the requirement of all of these residues for helicase activity. We also demonstrate that the duplex unwinding defects correlate with an inability of certain mutant proteins to translocate effectively on ssDNA. Moreover, the results define an essential triad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself.DNA-protein interactions ͉ motor proteins ͉ DNA translocation ͉ mutagenesis

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542followed by conversion to the Grignard reagent and reaction with propargyl bromide (procedure B) gave 9a.1,2Heptadiene-6,6-dz (9b) was prepared analogously to 9a starting with bromoethane-I, I -d2.3,CHeptadiene (1 l).32 Reaction of ethylmagnesium bromide with 3-bromo-1-pentyne (procedure B) gave 11. Abstract: A combination of chemical degradation and spectral data has established the structure of nogalamycin to be that Journal of the American Chemical Society / 99:2 / January 19,1977 shown in 1.

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