No abstract

Background:Competitors of LEDGF binding to HIV-1 integrase could prevent targeted integration to chromatin. Results: LEDGF competitors like tBPQAs were also found to inhibit integrase enzyme activity by preventing proper integraseviral DNA assembly. Conclusion: tBPQAs are allosteric inhibitors of integrase with a dual mode of action. Significance: Interference with two distinct steps of integration through the same binding site represents a new antiviral paradigm.

HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly.

E. coli UvrD is an SF1 helicase involved in several DNA metabolic processes. Although a UvrD dimer is needed for helicase activity, a monomer can translocate with 3' to 5' directionality along single-stranded DNA, and this ATP-dependent translocation is likely involved in RecA displacement. In order to understand how the monomeric translocase functions, we have combined fluorescence stopped-flow kinetic methods with recently developed analysis methods to determine the kinetic mechanism, including ATP coupling stoichiometry, for UvrD monomer translocation along ssDNA. Our results suggest that the macroscopic rate of UvrD monomer translocation is not limited by each ATPase cycle but rather by a slow step (pause) in each translocation cycle that occurs after four to five rapid 1 nt translocation steps, with each rapid step coupled to hydrolysis of one ATP. These results suggest a nonuniform stepping mechanism that differs from either a Brownian motor or previous structure-based inchworm mechanisms.

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 ...

Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA bound proteins, and basic properties like the elementary step size remain controversial. Using single molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single stranded loop. Static disorder limited previous ensemble studies of PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations suggesting a mode of action for eliminating potentially deleterious recombination intermediates.

Yeast Mec1/Ddc2 protein kinase, the ortholog of human ATR/ATRIP, plays a central role in the DNA damage checkpoint. The PCNA-like clamp Rad17/Mec3/Ddc1 (the 9-1-1 complex in human) and its loader Rad24-RFC are also essential components of this signal transduction pathway. Here we have studied the role of the clamp in regulating Mec1, and we delineate how the signal generated by DNA lesions is transduced to the Rad53 effector kinase. The checkpoint clamp greatly activates the kinase activity of Mec1, but only if the clamp is appropriately loaded upon partial duplex DNA. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, and the Rpa1 and Rpa2 subunits of RPA. Phosphorylation of Rad53, and of human PHAS-1, a nonspecific target, also requires a properly loaded clamp. Phosphorylation and binding studies with individual clamp subunits indicate that the Ddc1 subunit mediates the functional interactions with Mec1.

In bacteria, promoter recognition depends on the RNA polymerase sigma subunit, which combines with the catalytically proficient RNA polymerase core to form the holoenzyme. The major class of bacterial promoters is defined by two conserved elements (the -10 and -35 elements, which are 10 and 35 nucleotides upstream of the initiation point, respectively) that are contacted by sigma in the holoenzyme. We show that recognition of promoters of this class depends on the "flexible flap" domain of the RNA polymerase beta subunit. The flap interacts with conserved region 4 of sigma and triggers a conformational change that moves region 4 into the correct position for interaction with the -35 element. Because the flexible flap is evolutionarily conserved, this domain may facilitate promoter recognition by specificity factors in eukaryotes as well.