Ribonucleotide Misincorporation and Reverse Transcriptase Activities of Plasmodium falciparum Apicoplast DNA Polymerase.

Plasmodium falciparumis the most common and harmful causative agent of malaria worldwide. As a member of the phylum Apicomplexa, P. falciparum is characterized by the presence of a unique and essential organelle called the apicoplast. Reminiscent of an algal chloroplast, the apicoplast possesses its own genome, which is maintained by a single apicoplast DNA polymerase (apPol). Ribonucleotides misincorporated into the genome are among the most common lesions encountered by DNA polymerases, and the ability to replicate past these lesions varies widely among characterized enzymes. Here, we have investigated the ribonucleotide (rNTP) misincorporation frequency of apPol and determined its reverse transcriptase (RT) activity across templating ribonucleotides. Pre-steady-state kinetic experiments indicate that apPol does not have an unusually high discrimination between deoxy and ribonucleotides, with frequencies ranging between 104 and 106 depending on the identity of the ribonucleotide. Once incorporated into its template, apPol can replicate across ribonucleotides using its RT activity, but extension of a deoxynucleotide basepaired with the ribonucleotide is slow relative to a canonical basepair. Exonuclease assays indicate that apPol proofreads ribonucleotides an order of magnitude faster than extension, suggesting that most, but not all, misincorporated ribonucleotides will be excised. Although the components have not been identified, ribonucleotide excision repair or other tolerance mechanisms may exist in the P. falciparum apicoplast, and more targeted proteomic efforts will be needed to elucidate them.

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Polymerase Domain Insertion That Confers Dominant Exonuclease Activity.

Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme, remains enigmatic. De novo modeling of POLIB suggested a structure that is divergent from all other Family A polymerases, in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3'-5' exonucleases. Here we demonstrate recombinant POLIB 3'-5' exonuclease prefers DNA vs RNA substrates and degrades single- and double-stranded DNA nonprocessively. Exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB extends an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborates the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The enzymatic properties of POLIB suggest this paralog is not a replicative kDNA polymerase, and the noncanonical polymerase domain provides another example of exquisite diversity among DNA polymerases for specialized function.

Promising antimalarials targeting apicoplast DNA polymerase from Plasmodium falciparum.

Malaria is caused by the parasite Plasmodium falciparum, which contains an essential non-photosynthetic plastid called the apicoplast. A single DNA polymerase, apPOL, is targeted to the apicoplast, where it replicates and repairs the genome. apPOL has no direct orthologs in mammals and is considered a promising drug target for the treatment and/or prevention of malaria. We previously reported screening the Malaria Box to identify MMV666123 as an inhibitor of apPOL. Herein we extend our studies and report structure-activity relationships for MMV666123 and identify key structural motifs necessary for inhibition. Although attempts to crystallize apPOL with the inhibitor were not fruitful, kinetic analysis and crystal structure determinations of WT and mutant apo-enzymes, facilitated model building and provided insights into the putative inhibitor binding site. Our results validate apPOL as an antimalarial target and provide an avenue for the design of high potency, specific inhibitors of apPOL and other A-family DNA polymerases.

Discovery of small molecule inhibitors of Plasmodium falciparum apicoplast DNA polymerase.

Malaria is caused by infection with protozoan parasites of the Plasmodium genus, which is part of the phylum Apicomplexa. Most organisms in this phylum contain a relic plastid called the apicoplast. The apicoplast genome is replicated by a single DNA polymerase (apPOL), which is an attractive target for anti-malarial drugs. We screened small-molecule libraries (206,504 compounds) using a fluorescence-based high-throughput DNA polymerase assay. Dose/response analysis and counter-screening identified 186 specific apPOL inhibitors. Toxicity screening against human HepaRG human cells removed 84 compounds and the remaining were subjected to parasite killing assays using chloroquine resistant P. falciparum parasites. Nine compounds were potent inhibitors of parasite growth and may serve as lead compounds in efforts to discover novel malaria drugs.

Functional evaluation of the C-terminal region of bacteriophage T4 Rad50.

Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that participates in the processing of DNA ends for recombination-dependent DNA repair. Crystal and high-resolution cryo-EM structures of Rad50 have revealed DNA binding sites near the dimer interface of Rad50 opposite of Mre11, and near the base of the coiled-coils that extend out from the globular head domain. An analysis of T4-Rad50 using sequenced-based algorithms to identify DNA binding residues predicts that a conserved region of positively charged residues near the C-terminus, distal to the observed binding sites, interacts with DNA. Mutant proteins were generated to test this prediction and their enzymatic and DNA binding activities were evaluated. Consistent with the predictions, the Rad50 C-terminal mutants had reduced affinity for DNA as measured by Rad50 equilibrium DNA binding assays and an increased Km-DNA as determined in MR complex nuclease assays. Moreover, the allosteric activation of ATP hydrolysis activity due to DNA binding was substantially reduced, suggesting that these residues may be involved in the communication between the DNA and ATP binding sites.

Kinetic Analysis of the Exonuclease Activity of the Bacteriophage T4 Mre11-Rad50 Complex.

Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that is responsible for host genome degradation and the processing of DNA ends for recombination-dependent DNA repair. In this chapter, we describe the ensemble methods currently employed by our laboratory to characterize the exonuclease activity of the T4 MR complex. DNA exonucleases play a vital role in maintaining the integrity of DNA through their participation in DNA repair pathways and as proofreaders for DNA polymerases. Methods for quantifying the general features of the exonuclease, and for determining steady-state kinetic parameters (Km, kcat), the polarity of exonuclease activity, and processivity are presented. These methods should be applicable to all DNA exonucleases, and to some extent endonucleases.

Crystal Structure of the Apicoplast DNA Polymerase from Plasmodium falciparum: The First Look at a Plastidic A-Family DNA Polymerase.

Plasmodium falciparum, the primary cause of malaria, contains a non-photosynthetic plastid called the apicoplast. The apicoplast exists in most members of the phylum Apicomplexa and has its own genome along with organelle-specific enzymes for its replication. The only DNA polymerase found in the apicoplast (apPOL) was putatively acquired through horizontal gene transfer from a bacteriophage and is classified as an atypical A-family polymerase. Here, we present its crystal structure at a resolution of 2.9Å. P. falciparum apPOL, the first structural representative of a plastidic A-family polymerase, diverges from typical A-family members in two of three previously identified signature motifs and in a region not implicated by sequence. Moreover, apPOL has an additional N-terminal subdomain, the absence of which severely diminishes its 3' to 5' exonuclease activity. A compound known to be toxic to Plasmodium is a potent inhibitor of apPOL, suggesting that apPOL is a viable drug target. The structure provides new insights into the structural diversity of A-family polymerases and may facilitate structurally guided antimalarial drug design.

A network of allosterically coupled residues in the bacteriophage T4 Mre11-Rad50 complex.

The Mre11-Rad50 (MR) protein complex, made up of a nuclease and ATPase, respectively, is involved in the processing of double-strand breaks as part of an intricate mechanism for their repair. Although it is clear that the MR complex is subject to allosteric regulation and that there is communication between the nuclease and ATPase active sites, the underlying mechanisms are poorly understood. We performed statistical coupling analysis on Mre11 and Rad50 to predict linked residues based on their evolutionary correlation. This analysis predicted a coevolving sector of six residues that may be allosterically coupled. The prediction was tested using double-mutant cycle analysis of nuclease and ATPase activity. The results indicate that a tyrosine residue located near the active site of Mre11 is allosterically coupled to several Rad50 residues located over 40 Å away. This allosteric coupling may be the basis for the reciprocal regulation of the ATPase and nuclease activities of the complex.

Replication and maintenance of the Plasmodium falciparum apicoplast genome.

Members of the phylum Apicomplexa are responsible for many devastating diseases including malaria (Plasmodium spp.), toxoplasmosis (Toxoplasma gondii), babesiosis (Babesia bovis), and cyclosporiasis (Cyclospora cayetanensis). Most Apicomplexans contain a unique and essential organelle called the apicoplast. Derived from an ancient chloroplast, the apicoplast replicates and maintains a 35 kilobase (kb) circular genome. Due to its essential nature within the parasite, drugs targeted to proteins involved in DNA replication and repair of the apicoplast should be potent and specific. This review summarizes the current knowledge surrounding the replication and repair of the Plasmodium falciparum apicoplast genome and identifies several putative proteins involved in replication and repair pathways.

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain.

Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn(2+) cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn(2+) is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision.

Crystallization and preliminary X-ray analysis of the Plasmodium falciparum apicoplast DNA polymerase.

Infection by the parasite Plasmodium falciparum is the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment of Escherichia coli DNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystallization of and preliminary X-ray diffraction data from crystals of P. falciparum apPOL are reported. Data complete to 3.5 Šresolution were collected from a single crystal (2 × 2 × 5 µm) using a 5 µm beam. The space group P6522 (unit-cell parameters a = b = 141.8, c = 149.7 Å, α = ß = 90, γ = 120°) was confirmed by molecular replacement. Refinement is in progress.

Autoinhibition of bacteriophage T4 Mre11 by its C-terminal domain.

Mre11 and Rad50 form a stable complex (MR) and work cooperatively in repairing DNA double strand breaks. In the bacteriophage T4, Rad50 (gene product 46) enhances the nuclease activity of Mre11 (gene product 47), and Mre11 and DNA in combination stimulate the ATPase activity of Rad50. The structural basis for the cross-activation of the MR complex has been elusive. Various crystal structures of the MR complex display limited protein-protein interfaces that mainly exist between the C terminus of Mre11 and the coiled-coil domain of Rad50. To test the role of the C-terminal Rad50 binding domain (RBD) in Mre11 activation, we constructed a series of C-terminal deletions and mutations in bacteriophage T4 Mre11. Deletion of the RBD in Mre11 eliminates Rad50 binding but only has moderate effect on its intrinsic nuclease activity; however, the additional deletion of the highly acidic flexible linker that lies between RBD and the main body of Mre11 increases the nuclease activity of Mre11 by 20-fold. Replacement of the acidic residues in the flexible linker with alanine elevates the Mre11 activity to the level of the MR complex when combined with deletion of RBD. Nuclease activity kinetics indicate that Rad50 association and deletion of the C terminus of Mre11 both enhance DNA substrate binding. Additionally, a short peptide that contains the flexible linker and RBD of Mre11 acts as an inhibitor of Mre11 nuclease activity. These results support a model where the Mre11 RBD and linker domain act as an autoinhibitory domain when not in complex with Rad50. Complex formation with Rad50 alleviates this inhibition due to the tight association of the RBD and the Rad50 coiled-coil.

Catalytic mechanism of bacteriophage T4 Rad50 ATP hydrolysis.

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH-rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites.

A High-Throughput Assay to Identify Inhibitors of the Apicoplast DNA Polymerase from Plasmodium falciparum.

Infection by Plasmodium falciparum is the leading cause of malaria in humans. The parasite contains a unique and essential plastid-like organelle called the apicoplast that, similar to the mitochondria and chloroplast, houses its own genome that must undergo replication and repair. The putative apicoplast replicative DNA polymerase, POM1, has no direct orthologs in mammals, making the P. falciparum POM1 an attractive antimalarial drug target. Here, we report on a fluorescent high-throughput DNA polymerase assay that relies on the ability of POM1 to perform strand-displacement synthesis through the stem of a DNA hairpin substrate, thereby separating a Cy3 dye from a quencher. Assay-validation experiments were performed using 384-well plates and resulted in a signal window of 7.90 and aZ' factor of 0.71. A pilot screen of a 2880-compound library identified 62 possible inhibitors that cause more than 50% inhibition of polymerase activity. The simplicity and statistical robustness of the assay suggest it is well suited for the screening of novel apicoplast polymerase inhibitors that may serve as lead compounds in antimalarial drug-discovery efforts.

Fidelity, mismatch extension, and proofreading activity of the Plasmodium falciparum apicoplast DNA polymerase.

Plasmodium falciparum, a parasitic organism and one of the causative agents of malaria, contains an unusual organelle called the apicoplast. The apicoplast is a nonphotosynthetic plastid responsible for supplying the parasite with isoprenoid units and is therefore indispensable. Like mitochondria and the chloroplast, the apicoplast contains its own genome and harbors the enzymes responsible for its replication. In this report, we determine the relative probabilities of nucleotide misincorporation by the apicoplast polymerase (apPOL), examine the kinetics and sequence dependence of mismatch extension, and determine the rates of mismatch removal by the 3' to 5' proofreading activity of the DNA polymerase. While the intrinsic polymerase fidelity varies by >50-fold for the 12 possible nucleotide misincorporations, the most dominant selection step for overall polymerase fidelity is conducted at the level of mismatch extension, which varies by >350-fold. The efficiency of mismatch extension depends on both the nature of the DNA mismatch and the templating base. The proofreading activity of the 12 possible mismatches varies <3-fold. The data for these three determinants of polymerase-induced mutations indicate that the overall mutation frequency of apPOL is highly dependent on both the intrinsic fidelity of the polymerase and the identity of the template surrounding the potential mismatch.

A clamp-like biohybrid catalyst for DNA oxidation.

In processive catalysis, a catalyst binds to a substrate and remains bound as it performs several consecutive reactions, as exemplified by DNA polymerases. Processivity is essential in nature and is often mediated by a clamp-like structure that physically tethers the catalyst to its (polymeric) template. In the case of the bacteriophage T4 replisome, a dedicated clamp protein acts as a processivity mediator by encircling DNA and subsequently recruiting its polymerase. Here we use this DNA-binding protein to construct a biohybrid catalyst. Conjugation of the clamp protein to a chemical catalyst with sequence-specific oxidation behaviour formed a catalytic clamp that can be loaded onto a DNA plasmid. The catalytic activity of the biohybrid catalyst was visualized using a procedure based on an atomic force microscopy method that detects and spatially locates oxidized sites in DNA. Varying the experimental conditions enabled switching between processive and distributive catalysis and influencing the sliding direction of this rotaxane-like catalyst.

Coordination and processing of DNA ends during double-strand break repair: the role of the bacteriophage T4 Mre11/Rad50 (MR) complex.

The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of double-strand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections.

Interaction of T4 UvsW helicase and single-stranded DNA binding protein gp32 through its carboxy-terminal acidic tail.

Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA (ssDNA) binding protein gp32 is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW-catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy-terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions.

Disruption of the bacteriophage T4 Mre11 dimer interface reveals a two-state mechanism for exonuclease activity.

The Mre11-Rad50 (MR) complex is a central player in DNA repair and is implicated in the processing of DNA ends caused by double strand breaks. Recent crystal structures of the MR complex suggest that several conformational rearrangements occur during its ATP hydrolysis cycle. A comparison of the Mre11 dimer interface from these structures suggests that the interface is dynamic in nature and may adopt several different arrangements. To probe the functional significance of the Mre11 dimer interface, we have generated and characterized a dimer disruption Mre11 mutant (L101D-Mre11). Although L101D-Mre11 binds to Rad50 and dsDNA with affinity comparable with the wild-type enzyme, it does not activate the ATP hydrolysis activity of Rad50, suggesting that the allosteric communication between Mre11 and Rad50 has been interrupted. Additionally, the dsDNA exonuclease activity of the L101D-MR complex has been reduced by 10-fold under conditions where processive exonuclease activity is required. However, we unexpectedly found that under steady state conditions, the nuclease activity of the L101D-MR complex is significantly greater than that of the wild-type complex. Based on steady state and single-turnover nuclease assays, we have assigned the rate-determining step of the steady state nuclease reaction to be the productive assembly of the complex at the dsDNA end. Together, our data suggest that the Mre11 dimer interface adopts at least two different states during the exonuclease reaction.

Functional evaluation of bacteriophage T4 Rad50 signature motif residues.

The repair of DNA double-strand breaks (DSBs) is essential to maintaining the integrity of the genome, and organisms have evolved a conserved mechanism to facilitate their repair. In eukaryotes, archaea, and some bacteriophage, a complex made up of Mre11 and Rad50 (MR complex), which are a nuclease and ATPase, respectively, is involved in the initial processing of DSBs. Rad50 is a member of the ATP Binding Cassette (ABC) protein superfamily, the members of which contain an important Signature motif that acts in trans to complete the dimeric ATP binding site. To explore the functional relevance of this motif, four of its five residues were mutated in bacteriophage T4 Rad50, and their respective ATPase and nuclease activities were evaluated. The mutations reveal the functional roles of the Signature motif in ATP binding, hydrolysis, and cooperativity. In several mutants, the degree of DNA activation of ATP hydrolysis activity is reduced, indicating that the Signature motif is involved in allosteric signal transmission between the DNA and ATP binding sites of the MR complex. ATP hydrolysis is not required for nuclease activity when the probe is near the beginning of the DNA substrate; however, when an internal probe is used, decreases in ATPase activity have substantial effects on nuclease activity, suggesting that ATP hydrolysis is involved in translocation of the complex. Unexpectedly, the ATP hydrolysis and nuclease activities are not directly correlated with each other, and each mutation appears to differentially affect the exonuclease activity of Mre11.