High-Throughput Screening to Identify Inhibitors of SSB-Protein Interactions.

The bacterial single-stranded DNA-binding protein (SSB) uses an acidic C-terminal tail to interact with over a dozen proteins, acting as a genome maintenance hub. These SSB-protein interactions are essential, as mutations to the C-terminal tail that disrupt these interactions are lethal in Escherichia coli. While the roles of individual SSB-protein interactions have been dissected with mutational studies, small-molecule inhibitors of these interactions could serve as valuable research tools and have potential as novel antimicrobial agents. This chapter describes a high-throughput screening campaign used to identify inhibitors of SSB-protein interactions. A screen targeting the PriA-SSB interface from Klebsiella pneumoniae is presented as an example, but the methods may be adapted to target nearly any SSB interaction.

Hepatitis C Virus Helicase Binding Activity Monitored through Site-Specific Labeling Using an Expanded Genetic Code.

The mechanism of unwinding catalyzed by the hepatitis C virus nonstructural protein 3 helicase (NS3h) has been a subject of considerable interest, with NS3h serving as a prototypical enzyme in the study of helicase function. Recent studies support an ATP-fueled, inchworm-like stepping of NS3h on the nucleic acid that would result in the displacement of the complementary strand of the duplex during unwinding. Here, we describe the screening of a site of incorporation of an unnatural amino acid in NS3h for fluorescent labeling of the enzyme to be used in single-molecule Förster resonance energy transfer (FRET) experiments. From the nine potential sites identified in NS3h for incorporation of the unnatural amino acid, only one allowed for expression and fluorescent labeling of the recombinant protein. Incorporation of the unnatural amino acid was confirmed via bulk assays to not interfere with unwinding activity of the helicase. Binding to four different dsDNA sequences bearing a ssDNA overhang segment of varying length (either minimal 6 or 7 base length overhang to ensure binding or a long 24 base overhang) and sequence was recorded with the new NS3h construct at the single-molecule level. Single-molecule fluorescence displayed time intervals with anticorrelated donor and acceptor emission fluctuations associated with protein binding to the substrates. An apparent FRET value was estimated from the binding events showing a single FRET value of ∼0.8 for the 6-7 base overhangs. A smaller mean value and a broad distribution was in turn recorded for the long ssDNA overhang, consistent with NS3h exploring a larger physical space while bound to the DNA construct. Notably, intervals where NS3h binding was recorded were exhibited at time periods where the acceptor dye reversibly bleached. Protein induced fluorescence intensity enhancement in the donor channel became apparent at these intervals. Overall, the site-specific fluorescent labeling of NS3h reported here provides a powerful tool for future studies to monitor the dynamics of enzyme translocation during unwinding by single-molecule FRET.

[DYNAMICS AND MECHANISMS OF INTERACTION OF HETERO-HEXAMERIC TIP49a/b COMPLEXES WITH DS-DNA].

Evolutionary conserved TIP49a and TIP49b ATPases belong to the AAA+ superfamily of DNA-dependent ATPases that are involved in many cellular processes such as chromatin remodeling, regulation of transcription and cell division during mitosis, the maintenance of genome stability, snoRNP biogenesis, and participate in the formation of active form of telomerase. These proteins are involved in the complex networks of protein-protein interactions and, in spite of high structural similarity, in some cases, can perform opposite functions. Despite of the variety of their different activities, the exact mechanisms of action of TIP49a and TIP49b are still poorly understood. In this paper, by means of molecular docking approaches we first modeled the structures of hetero-hexameric TIP49 complexes with short ds-DNA fragments (20 base pairs with different GC content) within the central channel of hexameric ring. Using molecular dynamics simulations in the periodic water box (MD) we investigated conformational dynamics and mechanisms of DNA unwinding activity of these proteins. We shown that: a) the interaction between the positively charged protein loops and DNA within the central channel of protein ring leads to the partial unwinding of the DNA helix; b) DNA unwinding occurs only in the region within the protein ring, while the terminal parts of DNA outside the protein complex remain in its initial b-form conformation; c) the presence of ATP in the active sites of protein complex affects both the dynamics and the structure of DNA, leading to the breakage of some complementary bonds in AT-rich DNA sequences.

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3.

Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.

The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis.

The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase.

TWINKLE is a nucleus-encoded human mitochondrial (mt)DNA helicase. Point mutations in TWINKLE are associated with heritable neuromuscular diseases characterized by deletions in the mtDNA. To understand the biochemical basis of these diseases, it is important to define the roles of TWINKLE in mtDNA metabolism by studying its enzymatic activities. To this end, we purified native TWINKLE from Escherichia coli. The recombinant TWINKLE assembles into hexamers and higher oligomers, and addition of MgUTP stabilizes hexamers over higher oligomers. Probing into the DNA unwinding activity, we discovered that the efficiency of unwinding is greatly enhanced in the presence of a heterologous single strand-binding protein or a single-stranded (ss) DNA that is complementary to the unwound strand. We show that TWINKLE, although a helicase, has an antagonistic activity of annealing two complementary ssDNAs that interferes with unwinding in the absence of gp2.5 or ssDNA trap. Furthermore, only ssDNA and not double-stranded (ds)DNA competitively inhibits the annealing activity, although both DNAs bind with high affinities. This implies that dsDNA binds to a site that is distinct from the ssDNA-binding site that promotes annealing. Fluorescence anisotropy competition binding experiments suggest that TWINKLE has more than one ssDNA-binding sites, and we speculate that a surface-exposed ssDNA-specific site is involved in catalyzing DNA annealing. We propose that the strand annealing activity of TWINKLE may play a role in recombination-mediated replication initiation found in the mitochondria of mammalian brain and heart or in replication fork regression during repair of damaged DNA replication forks.

ATP-induced helicase slippage reveals highly coordinated subunits.

Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. Bacteriophage T7 gene product 4 is a model hexameric helicase that has been observed to use dTTP, but not ATP, to unwind double-stranded (ds)DNA as it translocates from 5' to 3' along single-stranded (ss)DNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective maximum rates V(max), Michaelis constants K(M) and concentrations. In contrast, processivity does not follow a simple competitive behaviour and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

XPB, a subunit of TFIIH, is a target of the natural product triptolide.

Triptolide (1) is a structurally unique diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities. Its molecular mechanism of action, however, has remained largely elusive to date. We report that triptolide covalently binds to human XPB (also known as ERCC3), a subunit of the transcription factor TFIIH, and inhibits its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II-mediated transcription and likely nucleotide excision repair. The identification of XPB as the target of triptolide accounts for the majority of the known biological activities of triptolide. These findings also suggest that triptolide can serve as a new molecular probe for studying transcription and, potentially, as a new type of anticancer agent through inhibition of the ATPase activity of XPB.

Running in reverse: the structural basis for translocation polarity in hexameric helicases.

Hexameric helicases couple ATP hydrolysis to processive separation of nucleic acid duplexes, a process critical for gene expression, DNA replication, and repair. All hexameric helicases fall into two families with opposing translocation polarities: the 3'-->5' AAA+ and 5'-->3' RecA-like enzymes. To understand how a RecA-like hexameric helicase engages and translocates along substrate, we determined the structure of the E. coli Rho transcription termination factor bound to RNA and nucleotide. Interior nucleic acid-binding elements spiral around six bases of RNA in a manner unexpectedly reminiscent of an AAA+ helicase, the papillomavirus E1 protein. Four distinct ATP-binding states, representing potential catalytic intermediates, are coupled to RNA positioning through a complex allosteric network. Comparative studies with E1 suggest that RecA and AAA+ hexameric helicases use different portions of their chemomechanical cycle for translocating nucleic acid and track in opposite directions by reversing the firing order of ATPase sites around the hexameric ring. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

Structure and function of primase RepB' encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode.

For the initiation of DNA replication, dsDNA is unwound by helicases. Primases then recognize specific sequences on the template DNA strands and synthesize complementary oligonucleotide primers that are elongated by DNA polymerases in leading- and lagging-strand mode. The bacterial plasmid RSF1010 provides a model for the initiation of DNA replication, because it encodes the smallest known primase RepB' (35.9 kDa), features only 1 single-stranded primase initiation site on each strand (ssiA and ssiB, each 40 nt long with 5'- and 3'-terminal 6 and 13 single-stranded nucleotides, respectively, and nucleotides 7-27 forming a hairpin), and is replicated exclusively in leading strand mode. We present the crystal structure of full-length dumbbell-shaped RepB' consisting of an N-terminal catalytic domain separated by a long alpha-helix and tether from the C-terminal helix-bundle domain and the structure of the catalytic domain in a specific complex with the 6 5'-terminal single-stranded nucleotides and the C7-G27 base pair of ssiA, its single-stranded 3'-terminus being deleted. The catalytic domains of RepB' and the archaeal/eukaryotic family of Pri-type primases share a common fold with conserved catalytic amino acids, but RepB' lacks the zinc-binding motif typical of the Pri-type primases. According to complementation studies the catalytic domain shows primase activity only in the presence of the helix-bundle domain. Primases that are highly homologous to RepB' are encoded by broad-host-range IncQ and IncQ-like plasmids that share primase initiation sites ssiA and ssiB and high sequence identity with RSF1010.

Structural modeling of the NS 3 helicase of Tick-borne encephalitis virus and their virtual screening of potent drugs using molecular docking.

Homology modeling of NS3 helicase in Tick-borne encephalitis virus using known protein crystal structure was done. Its 3-D structure was evaluated and validated using PROCHECK comprising amino acid residues in favored region of Ramachandran plot. Helicase forms a large family of proteins which ubiquitously distributes in wide variety of organisms. It plays crucial role in transcription and replication of single-stranded viral RNA genomes. Consequently, NS3 represents an interesting target for the development of specific antiviral inhibitors. Several helicase inhibiting effective drugs and analogs were selected and the active amino acid residues were targeted. Levovirin, Ribamidine and Ribavirin were found more potent to inhibit TBEV on the basis of robust binding affinity between protein-drug interactions. This finding may help to understand the nature of helicase and development of specific anti-TBEV therapies.

Non-hexameric DNA helicases and translocases: mechanisms and regulation.

Helicases and nucleic acid translocases are motor proteins that have essential roles in nearly all aspects of nucleic acid metabolism, ranging from DNA replication to chromatin remodelling. Fuelled by the binding and hydrolysis of nucleoside triphosphates, helicases move along nucleic acid filaments and separate double-stranded DNA into their complementary single strands. Recent evidence indicates that the ability to simply translocate along single-stranded DNA is, in many cases, insufficient for helicase activity. For some of these enzymes, self assembly and/or interactions with accessory proteins seem to regulate their translocase and helicase activities.

Discovery of novel DNA gyrase inhibitors by high-throughput virtual screening.

The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains.

Biochemical, biophysical, and proteomic approaches to study DNA helicases.

Helicases are a family of enzymes that play an essential role in nearly all DNA metabolic processes, catalyzing the transient opening of DNA duplexes. These motor proteins couple the chemical energy of ATP binding and hydrolysis to the separation of the complementary strands of a DNA or RNA duplex substrate. A full understanding of their mechanism of DNA unwinding can be achieved only through careful investigation of the thermodynamic and kinetic parameters that control this ATP-driven process, as well as through analysis of the helicases' tertiary and quaternary structures associated with nucleic acids and/or nucleotide recognition. This review describes the various biochemical, biophysical, and, more recently, proteomic techniques that have been developed to shed light on the still controversial, and in some aspects elusive, helicase-catalyzed mechanism of DNA unwinding.

DNA unwinding assay using streptavidin-bound oligonucleotides.

BACKGROUND: Helicases play essential roles in many cellular processes including replication, transcription and translation. Most helicases translocate along one strand of the duplex while displacing the complementary strand (of either DNA or RNA). Thus, helicases have directionality. They move along nucleic acids in either the 3'--> 5' or 5'--> 3' direction. The directionality of helicases with low activity or of those that cannot initiate duplex unwinding from a substrate that contains only one single-stranded overhang region is difficult to determine. RESULTS: An improved assay to determine helicase directionality was developed that uses a substrate containing biotinylated oligonucleotides. As a proof of concept, it was shown that the substrates substantially improve helicase activity and directionality determination for several DNA helicases in comparison to more traditional substrates. In addition, a universal substrate that can be used to determine the directionality of both 3'--> 5' and 5'--> 3' helicases was developed. CONCLUSION: It is shown here that the use of a biotin-streptavidin complex as a helicase substrate improves helicase activity and the determination of helicase directionality. The method described is simpler that the currently available techniques.

Biochemical assays for the characterization of DNA helicases.

Helicases are ubiquitous enzymes that disrupt complementary strands of duplex nucleic acid in a reaction dependent on nucleoside-5'-triphosphate hydrolysis. Helicases are implicated in the metabolism of DNA structures that are generated during replication, recombination, and DNA repair. Furthermore, an increasing number of helicases have been linked to genomic instability and human disease. With the growing interest in helicase mechanism and function, we have set out to describe some basic protocols for biochemical characterization of DNA helicases. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding activity of DNA helicases are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of a given helicase, which would serve as a platform for further investigation of its molecular and cellular functions.

Hepatocellular carcinoma caused by iron overload: a possible mechanism of direct hepatocarcinogenicity.

BACKGROUND/AIMS: Excess hepatic iron may be both directly and indirectly carcinogenic. The aim of this study was to determine if generation of reactive oxygen species and the resulting oxidative damage induced by free hepatic iron is directly hepatocarcinogenic. METHODS: Sixty male Wistar albino rats were iron-loaded by ferrocene supplementation of their diet. Biochemical parameters of oxidative damage and lipid peroxidation, DNA unwinding and strand breaks, and the Ames Mutagenesis Test were measured at 4 monthly intervals and correlated with the degree of hepatic iron overload, the presence of iron-free preneoplastic foci in the liver, and the development of hepatocellular carcinoma in comparison with 60 control rats. RESULTS: Levels of lipid hydroperoxides, malonaldehyde, 8-isoprostane and 8-hydroxy-2'-deoxyguanosine increased, reaching peak concentrations at 20-24 months, and correlating with an increase in the rate of DNA unwinding, strand breaks, and positive Ames Tests. Iron-free neoplastic foci became evident at 16 months and thereafter increased in number. Preneoplastic foci were present in five of eight rats remaining at 32 months and HCC had developed in one of the five. CONCLUSIONS: Our findings are compatible with the hypothesis that the direct hepatocarcinogenic effect of free iron is mediated by the generation of oxygen reactive species and oxidative damage that are mutagenic and carcinogenic.

A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex.

The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665-13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding.

Cold stress-induced pea DNA helicase 47 is homologous to eIF4A and inhibited by DNA-interacting ligands.

Helicases are ubiquitous molecular motor proteins that play important role in maintaining the genome integrity and thus involved in plant growth and development. Here, we report the cloning of cDNA (1.64 kb) and genomic DNA (2.2 kb) of cold stress-induced pea DNA helicase 47 (PDH47) and characterization of its encoded protein. It belongs to DEAD-box protein family and shows striking identity (93%) with tobacco eIF4A. The transcript was induced under cold (4 degrees C) stress. The purified PDH47 protein (47 kDa) contains ATP-/Mg2+-dependent DNA unwinding as well as DNA-/Mg2+-dependent ATPase activities. The ATPase activity of PDH47 is stimulated more by ssDNA as compared to dsDNA and RNA. The activities of PDH47 are inhibited by various DNA-interacting ligands such as nogalamycin, daunorubicin, ethidium bromide, mitoxantrone, actinomycin, and cisplatin with apparent Ki values ranging from 0.5 to 8.0 microM. Interestingly, netropsin and distamycin inhibited the helicase but not the ATPase activity. The inhibition might be due to the intercalation of inhibitors into duplex DNA, which can impede the translocation of the PDH47. This study should help in our better understanding of cold stress signaling and mechanism of DNA unwinding in plants.

Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human RECQ1.

RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability.