DNA helicases catalyze separation of double-helical DNA into its complementary single strands, a process essential for DNA replication, recombination, and repair. The Escherichia coli Rep protein, a superfamily 1 DNA helicase, functions in DNA replication restart and is required for replication of several bacteriophages. Monomers of Rep do not display helicase activity in vitro; in fact, DNA unwinding requires Rep dimerization. Here we show that removal of the 2B subdomain of Rep to form Rep⌬2B activates monomer helicase activity, albeit with limited processivity. Although both full length Rep and Rep⌬2B monomers can translocate with 3 to 5 directionality along single-stranded DNA, the 2B subdomain inhibits the helicase activity of full length Rep. This suggests an autoregulatory mechanism for Rep helicase, which may apply to other nonhexameric helicases, whereby helicase activity is regulated by the rotational conformational state of the 2B subdomain; formation of a Rep dimer may relieve autoinhibition by altering the 2B subdomain orientation.DNA unwinding ͉ kinetics ͉ replication ͉ translocation D NA helicases are a ubiquitous class of enzymes that use the binding and hydrolysis of nucleoside triphosphates to catalyze the separation of the DNA double helix into its complementary single strands. This process is essential for DNA replication, recombination, and repair (1-3), and defects in some DNA helicases are linked to human diseases (4-6). DNA helicases are classified into superfamilies based on their primary structure, with the majority belonging to superfamilies (SF)1 and SF2 (7). Some DNA helicases function as hexamers (8); others, such as the Escherichia coli SF1 helicases Rep (9) and UvrD (10) and the SF2 hepatitis C viral (HCV) NS3 helicase (11), function as dimers in vitro, whereas others, such as the SF1 phage T4 Dda helicase (12), show limited activity as monomers in vitro. The SF1 Bacillus stearothermophilus PcrA helicase has been proposed to function as a monomer (13), although this has not been demonstrated experimentally. Possible roles for oligomerization in helicase activity have been discussed (3,8,14).The E. coli Rep protein (673 amino acids), a 3Ј to 5Ј SF1 DNA helicase (3,14), is involved in replication restart (15) and is required for replication of some bacteriophages (16). Rep exists as a monomer in solution in the absence of DNA; however, in vitro, Rep monomers are inactive as helicases, and Rep dimerization is required for processive DNA unwinding (9, 17). E. coli Rep is structurally homologous (18) to B. stearothemophilus PcrA (19) and E. coli UvrD (S. Korolev, N. K. Maluf, T.M.L., and G. Waksman, unpublished results), both of which are also 3Ј to 5Ј SF1 DNA helicases. Rep monomer is composed of two domains (1 and 2), each with two subdomains (1A, 2A, 1B, and 2B) (Fig. 1). In the asymmetric unit of the Rep-(dT 16 ) crystal structure, two molecules of Rep are observed that differ in the orientation of the 2B subdomain (18). These two orientations (''open'' vs. ''closed'') differ by a ...

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The structural mechanism by which non-structural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 (SF2) helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3’-end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a “spring-loading” mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.

Diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitors (NNRTIs) have inherent flexibility, helping to maintain activity against a wide range of resistance mutations. Crystal structures were determined with wild-type and K103N HIV-1 reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278). These structures reveal a similar binding mode for TMC125 and TMC278, whether bound to wild-type or K103N RT. Comparison to previously published structures reveals differences in binding modes for TMC125 and differences in protein conformation for TMC278.

In plants and Plasmodium falciparum, the synthesis of phosphatidylcholine requires the conversion of phosphoethanolamine to phosphocholine by phosphoethanolamine methyltransferase (PEAMT). This pathway differs from the metabolic route of phosphatidylcholine synthesis used in mammals and, on the basis of bioinformatics, was postulated to function in the nematode Caenorhabditis elegans. Here we describe the cloning and biochemical characterization of a PEAMT from C. elegans (gene, pmt-2; protein, PMT-2). Although similar in size to the PEAMT from plants, which contain two tandem methyltransferase domains, PMT-2 retains only the C-terminal methyltransferase domain. RNA-mediated interference experiments in C. elegans show that PMT-2 is essential for worm viability and that choline supplementation rescues the RNAi-generated phenotype. Unlike the plant and Plasmodium PEAMT, which catalyze all three methylations in the pathway, PMT-2 catalyzes only the last two steps in the pathway, i.e., the methylation of phosphomonomethylethanolamine (P-MME) to phosphodimethylethanolamine (P-DME) and of P-DME to phosphocholine. Analysis of initial velocity patterns suggests a random sequential kinetic mechanism for PMT-2. Product inhibition by S-adenosylhomocysteine was competitive versus S-adenosylmethionine and noncompetitive versus P-DME, consistent with formation of a dead-end complex. Inhibition by phosphocholine was competitive versus each substrate. Fluorescence titrations show that all substrates and products bind to the free enzyme. The biochemical data are consistent with a random sequential kinetic mechanism for PMT-2. This work provides a kinetic basis for additional studies on the reaction mechanism of PEAMT. Our results indicate that nematodes also use the PEAMT pathway for phosphatidylcholine biosynthesis. If the essential role of PMT-2 in C. elegans is conserved in parasitic nematodes of mammals and plants, then inhibition of the PEAMT pathway may be a viable approach for targeting these parasites with compounds of medicinal or agronomic value.