Crystal structures of YwqE from Bacillus subtilis and CpsB from Streptococcus pneumoniae, unique metal-dependent tyrosine phosphatases

Unique metal-dependent protein tyrosine phosphatases that belong to the polymerase and histindinol phosphatase (PHP) family are present in Gram-positive bacteria. They are distinct from the Cys-based, low-molecular-weight phosphotyrosine protein phosphatases (LMPTPs). Two representative members of the PHP family tyrosine phosphatases are YwqE from Bacillus subtilis and CpsB from Streptococcus pneumoniae. YwqE is involved in polysaccharide biosynthesis, bacterial DNA metabolism, and DNA damage response in B. subtilis. CpsB regulates capsular polysaccharide biosynthesis via tyrosine dephosphorylation of CpsD, its cognate tyrosine kinase, in S. pneumoniae. To gain insights into the active site and possible conformational changes of the metal-dependent tyrosine phosphatases from Gram-positive bacteria, we have determined the crystal structures of B. subtilis YwqE (in both the apo form and the phosphate-bound form) and S. pneumoniae CpsB (in the sulfate-bound form). Comparisons of the three structures reveal conformational plasticity of two active site loops. Furthermore, in both structures of the phosphate-bound YwqE and the sulfate-bound CpsB, the phosphate (or sulfate) ion is bound to a cluster of three metal ions in the active site, thus providing insight into the pre-catalytic state.     

Mechanistic insights into the enzymatic activity and inhibition of the replicative polymerase exonuclease domain from Mycobacterium tuberculosis

DNA replication fidelity maintains low mutation rates in bacteria. The ε-subunit of a replisome generally acts as the main proofreader during replication, using its 3′–5′ exonuclease activity to excise misincorporated bases thereby maintaining faithful replication. In Mycobacterium tuberculosis (Mtb), however, the polymerase and histidinol phosphatase (PHP) domain of the DNA polymerase DnaE1 is the primary proofreader. This domain thus maintains low mutation rates during replication and is an attractive target for drug development. Even though the structures of DnaE polymerases are available from various organisms, including Mtb, the mechanism of exonuclease activity remains elusive. In this study, we sought to unravel the mechanism and also to identify scaffolds that can specifically inhibit the exonuclease activity. To gain insight into the mode of action, we also characterized the PHP domain of the Mtb error-prone polymerase DnaE2 which shares a nearly identical active site with DnaE1-PHP. Kinetic and mutational studies allowed us to identify the critical residue involved in catalysis. Combined inhibition and computational studies also revealed a specific mode of inhibition of DnaE1-PHP by nucleoside diphosphates. Thus, this study lays the foundation for the rational design of novel inhibitors which target the Mtb replicative proofreader.     

Streptococcus pneumoniae Capsule Biosynthesis Protein CpsB Is a Novel Manganese-Dependent Phosphotyrosine-Protein Phosphatase

The first four genes of the capsule locus (cps) of Streptococcus pneumoniae (cpsA to cpsD) are common to most serotypes. We have previously determined that CpsD is an autophosphorylating protein-tyrosine kinase, demonstrated that CpsC is required for CpsD tyrosine-phosphorylation, and shown that CpsB is required for dephosphorylation of CpsD. In the present study we show that CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase that belongs to the PHP (polymerase and histidinol phosphatase) family of phosphoesterases. We also show that an S. pneumoniae strain with point mutations in cpsB, affecting one of the conserved motifs of CpsB, is unencapsulated and appears to be morphologically identical to a strain in which the cpsB gene had been deleted.     

Crystal Structures of Wzb of Escherichia coli and CpsB of Streptococcus pneumoniae, Representatives of Two Families of Tyrosine Phosphatases that Regulate Capsule Assembly

Many Gram-positive and Gram-negative bacteria utilize polysaccharide surface layers called capsules to evade the immune system; consequently, the synthesis and export of the capsule are a potential therapeutic target. In Escherichia coli K-30, the integral membrane tyrosine autokinase Wzc and the cognate phosphatase Wzb have been shown to be key for both synthesis and assembly of capsular polysaccharides. In the Gram-positive bacterium Streptococcus pneumoniae, the CpsCD complex is analogous to Wzc and the phosphatase CpsB is the corresponding cognate phosphatase. The phosphatases are known to dephosphorylate their corresponding autokinases, yet despite their functional equivalence, they share no sequence homology. We present the structure of Wzb in complex with phosphate and high-resolution structures of apo-CpsB and a phosphate-complexed CpsB. We show that both proteins are active toward Wzc and thereby demonstrate that CpsB is not specific for CpsCD. CpsB is a novel enzyme and represents the first solved structure of a tyrosine phosphatase from a Gram-positive bacterium. Wzb and CpsB have completely different structures, suggesting that they must operate by very different mechanisms. Although the mechanism of Wzb can be inferred from previous studies, CpsB appears to have a tyrosine phosphatase mechanism not observed before. We propose a chemical mechanism for CpsB based on site-directed mutagenesis and structural data.     

DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3'-->5' exonuclease activity involved in radioresistance

Recently a family X DNA polymerase (PolXDr) was identified in the radioresistant bacterium Deinococcus radiodurans. Knockout cells show a delay in double-strand break repair (DSBR) and an increased sensitivity to gamma-irradiation. Here we show that PolXDr possesses 3'-->5' exonuclease activity that stops cutting close to a loop. PolXDr consists of a DNA polymerase X domain (PolXc) and a Polymerase and Histidinol Phosphatase (PHP) domain. Deletion of the PHP domain abolishes only the structure-modulated but not the canonical 3'-->5' exonuclease activity. Thus, the exonuclease resides in the PolXc domain, but the structure-specificity requires additionally the PHP domain. Mutation of two conserved glycines in the PolXc domain leads to a specific loss of the structure-modulated exonuclease activity but not the exonuclease activity in general. The PHP domain itself does not show any activity. PolXDr is the first family X DNA polymerase that harbours an exonuclease activity. The wild-type protein, the glycine mutant and the two domains were expressed separately in DeltapolXDr cells. The wild-type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of gamma-irradiated cells. Taken together our in vivo results suggest that both PolXDr domains play important roles in DSBR in D. radiodurans.     

Phosphoesterase domains associated with DNA polymerases of diverse origins.

Computer analysis of DNA polymerase protein sequences revealed previously unidentified conserved domains that belong to two distinct superfamilies of phosphoesterases. The alpha subunits of bacterial DNA polymerase III and two distinct family X DNA polymerases are shown to contain an N-terminal domain that defines a novel enzymatic superfamily, designated PHP, after polymerase and histidinol phosphatase. The predicted catalytic site of the PHP superfamily consists of four motifs containing conserved histidine residues that are likely to be involved in metal-dependent catalysis of phosphoester bond hydrolysis. The PHP domain is highly conserved in all bacterial polymerase III alpha subunits, but in proteobacteria and mycoplasmas, the conserved motifs are distorted, suggesting a loss of the enzymatic activity. Another conserved domain, found in the small subunits of archaeal DNA polymerase II and eukaryotic DNA polymerases alpha and delta, is shown to belong to the superfamily of calcineurin-like phospho-esterases, which unites a variety of phosphatases and nucleases. The conserved motifs required for phospho-esterase activity are intact in the archaeal DNA polymerase subunits, but are disrupted in their eukaryotic orthologs. A hypothesis is proposed that bacterial and archaeal replicative DNA polymerases possess intrinsic phosphatase activity that hydrolyzes the pyrophosphate released during nucleotide polymerization. As proposed previously, pyrophosphate hydrolysis may be necessary to drive the polymerization reaction forward. The phosphoesterase domains with disrupted catalytic motifs may assume an allosteric, regulatory function and/or bind other subunits of DNA polymerase holoenzymes. In these cases, the pyrophosphate may be hydrolyzed by a stand-alone phosphatase, and candidates for such a role were identified among bacterial PHP superfamily members.     

The Family X DNA Polymerase from Deinococcus radiodurans Adopts a Non-standard Extended Conformation

Deinococcus radiodurans is an extraordinarily radioresistant bacterium that is able to repair hundreds of radiation-induced double-stranded DNA breaks. One of the players in this pathway is an X family DNA polymerase (PolX_Dr). Deletion of PolX_Dr has been shown to decrease the rate of repair of double-stranded DNA breaks and increase cell sensitivity to gamma-rays. A 3′→5′ exonuclease activity that stops cutting close to DNA loops has also been demonstrated. The present crystal structure of PolX_Dr solved at 2.46-Å resolution reveals that PolX_Dr has a novel extended conformation in stark contrast to the closed “right hand” conformation commonly observed for DNA polymerases. This extended conformation is stabilized by the C-terminal PHP domain, whose putative nuclease active site is obstructed by its interaction with the polymerase domain. The overall conformation and the presence of non standard residues in the active site of the polymerase X domain makes PolX_Dr the founding member of a novel class of polymerases involved in DNA repair but whose detailed mode of action still remains enigmatic.DNA replication and repair are functions that are of vital importance for the maintenance of cellular life. These functions are carried out by various DNA replicating engines, most of them acting as multiprotein complexes. Deinococcus radiodurans, a Gram-positive bacterium, is characterized by an extraordinary resistance to ionizing radiation and desiccation. After radiation induced cutting of its 3.28-megabase genome into hundreds of small fragments, it is capable of reassembling it completely (1). Different hypotheses have been suggested to explain this radioresistance. A recently proposed mechanism involves the creation of long linear DNA intermediates by an extended synthesis-dependent strand annealing process, where overlapping chromosomal fragments are used both as primers and as templates for synthesis of complementary single strands (2). Recircularization of chromosomes would be assured by homologous recombination. Although DNA polymerase I is one of the main enzymes involved in this process, it was shown that other proteins affect double strand break repair efficiency in D. radiodurans. One of these is an X family DNA polymerase (PolX_Dr)⁵ (3). Cells devoid of PolX_Dr protein show increased sensitivity to γ-irradiation and a longer delay in the restoration of an intact genome after irradiation. It was therefore proposed that PolX_Dr has an important role in double strand break repair in D. radiodurans. The contribution of PolX_Dr may become essential for instance when damage gets too important or, alternatively, it may act in different repair pathways from polymerase I. Indeed, some of the X DNA polymerases, such as Saccharomyces cerevisiae Pol4 and human polymerase λ (4) have been proposed to play important roles in different DNA repair processes, including non-homologous end-joining (5). It was shown that PolX_Dr also has strong 3′→5′ exonuclease activity that is stimulated by Mn²⁺ (6). This activity is associated with proofreading mechanisms in other polymerase families and encoded by protein domains or subunits distinct from the polymerase catalytic domain (7). Curiously the exonuclease activity of PolX_Dr is modulated upon encounter of a stem-loop structure. The combination of both activities leads to the hypothesis that PolX_Dr might be involved in DNA repair, potentially non-homologous end-joining, by processing damaged DNA or repair intermediates, thus generating substrates for other repair proteins (6). Very recently an orthologue of PolX from Bacillus subtilis was characterized. It was shown that PolX_Bs is a template-directed DNA polymerase acting on DNA gaps with a downstream 5′ phosphate group, suggesting it may play a role in base excision repair (8).DNA polymerases all combine a catalytic palm domain, a thumb domain, binding double-stranded DNA, and a finger domain that fixes the incoming nucleotide. The polymerase domain of the X family belongs to the Polβ-like nucleotidyltransferase superfamily, sharing ∼25% amino acid identity with the DNA polymerase domains of Polλ, Pol4, and Polβ. PolX_Dr has a second domain at the C terminus called PHP, with strong sequence identity with the histidinol phosphatase involved in histidine transport in bacteria. Due to its similarity to histidinol phosphatase and the presence of a trinuclear zinc site, the PolX_Dr PHP domain is thought to function as phosphoesterase (9). In the context of DNA polymerases, this activity might be responsible for the degradation of pyrophosphate, thus driving the polymerization reaction, or contributes to a nuclease reaction that would be involved in proofreading the newly synthesized strand. The deletion of the PHP domain also had a negative effect on survival of γ-irradiated cells suggesting that this domain possesses a function in DNA repair. Unexpectedly, deletion of the PHP domain destroys structure modulated but not the general 3′→5′ exonuclease activity (6). No activity could be demonstrated for the PHP domain alone.In this report we present the crystal structure of PolX_Dr at 2.46-Å resolution. Surprisingly, PolX_Dr adopts a stretched out conformation instead of the commonly observed closed right hand conformation. In the active site of the polymerase catalytic domain, the two universally conserved aspartates are replaced by two glutamates, whereas the active site of the PHP domain is obstructed by its interaction with the polymerase domain.     

The DNA replication machine of a gram-positive organism

This report outlines the protein requirements and subunit organization of the DNA replication apparatus of Streptococcus pyogenes, a Gram-positive organism. Five proteins coordinate their actions to achieve rapid and processive DNA synthesis. These proteins are: the PolC DNA polymerase, tau, delta, delta', and beta. S. pyogenes dnaX encodes only the full-length tau, unlike the Escherichia coli system in which dnaX encodes two proteins, tau and gamma. The S. pyogenes tau binds PolC, but the interaction is not as firm as the corresponding interaction in E. coli, underlying the inability to purify a PolC holoenzyme from Gram-positive cells. The tau also binds the delta and delta' subunits to form a taudeltadelta' "clamp loader." PolC can assemble with taudeltadelta' to form a PolC.taudeltadelta' complex. After PolC.taudeltadelta' clamps beta to a primed site, it extends DNA 700 nucleotides/second in a highly processive fashion. Gram-positive cells contain a second DNA polymerase, encoded by dnaE, that has homology to the E. coli alpha subunit of E. coli DNA polymerase III. We show here that the S. pyogenes DnaE polymerase also functions with the beta clamp.     

Editing of misaligned 3'-termini by an intrinsic 3'-5' exonuclease activity residing in the PHP domain of a family X DNA polymerase

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolX(Bs)), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolX(Bs) possesses an intrinsic 3'-5' exonuclease activity specialized in resecting unannealed 3'-termini in a gapped DNA substrate. Biochemical analysis of a PolX(Bs) deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3'-5' exonuclease activity of PolX(Bs) resides in its PHP domain. Furthermore, site-directed mutagenesis of PolX(Bs) His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3'-termini resection by the 3'-5' exonuclease activity of PolX(Bs) in the DNA repair context are discussed.     

The N-terminal region of the bacterial DNA polymerase PolC features a pair of domains, both distantly related to domain V of the DNA polymerase III τ subunit

PolC is one of two essential replicative DNA polymerases in Bacillus subtilis and other Gram-positive bacteria. The 3D structure of PolC has recently been solved, yet it lacks the N-terminal region. For this PolC region of ～ 230 residues, both the structure and function are unknown. In the present study, using sensitive homology detection and comparative protein structure modeling, we identified, in this enigmatic region, two consecutive globular domains, PolC-NI and PolC-NII, which are followed by an apparently unstructured linker. Unexpectedly, we found that both domains are related to domain V of the τ subunit, which is part of the bacterial DNA polymerase III holoenzyme. Despite their common homology to τ, PolC-NI and PolC-NII exhibit very little sequence similarity to each other. This observation argues against simple tandem duplication within PolC as the origin of the two-domain structure. Using the derived structural models, we analyzed residue conservation and the surface properties of both PolC N-terminal domains. We detected a surface patch of positive electrostatic potential in PolC-NI and a hydrophobic surface patch in PolC-NII, suggesting their possible involvement in nucleic acid and protein binding, respectively. PolC is known to interact with the τ subunit, however, the region responsible for this interaction is unknown. We propose that the PolC N-terminus is involved in mediating the PolC-τ interaction and possibly also in binding DNA.     

Conserved interactions in the Staphylococcus aureus DNA PolC chromosome replication machine

The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, tau, is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli tau. S. aureus tau also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III.tau. We demonstrate that the extreme C-terminal residues of PolC bind to and function with beta clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus delta wrench of the clamp loader binds to E. coli beta. The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes beta clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous beta clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them.     

Crystal structure of a metal-dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis: Computational prediction and experimental validation of phosphoesterase activity

The crystal structures of an unliganded and adenosine 5′‐monophosphate (AMP) bound, metal‐dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis are reported at 2.4 and 1.94 Å, respectively. Functional characterization of this enzyme was guided by computational analysis and then confirmed by experiment. The structure consists of a polymerase and histidinol phosphatase (PHP, Pfam: PF02811) domain with a second domain (residues 105‐178) inserted in the middle of the PHP sequence. The insert domain functions in binding AMP, but the precise function and substrate specificity of this domain are unknown. Initial bioinformatics analyses yielded multiple potential functional leads, with most of them suggesting DNA polymerase or DNA replication activity. Phylogenetic analysis indicated a potential DNA polymerase function that was somewhat supported by global structural comparisons identifying the closest structural match to the alpha subunit of DNA polymerase III. However, several other functional predictions, including phosphoesterase, could not be excluded. Theoretical microscopic anomalous titration curve shapes, a computational method for the prediction of active sites from protein 3D structures, identified potential reactive residues in YP_910028.1. Further analysis of the predicted active site and local comparison with its closest structure matches strongly suggested phosphoesterase activity, which was confirmed experimentally. Primer extension assays on both normal and mismatched DNA show neither extension nor degradation and provide evidence that YP_910028.1 has neither DNA polymerase activity nor DNA‐proofreading activity. These results suggest that many of the sequence neighbors previously annotated as having DNA polymerase activity may actually be misannotated. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Bacterial replicases and related polymerases

Bacterial replicases are complex, tripartite replicative machines. They contain a polymerase, Pol III, a β(2) processivity factor and a DnaX complex ATPase that loads β(2) onto DNA and chaperones Pol III onto the newly loaded β(2). Many bacteria encode both a full length τ and a shorter γ form of DnaX by a variety of mechanisms. The polymerase catalytic subunit of Pol III, α, contains a PHP domain that not only binds to prototypical ? Mg(2+)-dependent exonuclease, but also contains a second Zn(2+)-dependent proofreading exonuclease, at least in some bacteria. Replication of the chromosomes of low GC Gram-positive bacteria require two Pol IIIs, one of which, DnaE, appears to extend RNA primers a only short distance before handing the product off to the major replicase, PolC. Other bacteria encode a second Pol III (ImuC) that apparently replaces Pol V, required for induced mutagenesis in E. coli. Approaches that permit simultaneous biochemical screening of all components of complex bacterial replicases promise inhibitors of specific protein targets and reaction stages.     

Pyrophosphate release acts as a kinetic checkpoint during high-fidelity DNA replication by the Staphylococcus aureus replicative polymerase PolC

Bacterial replication is a fast and accurate process, with the bulk of genome duplication being catalyzed by the α subunit of DNA polymerase III within the bacterial replisome. Structural and biochemical studies have elucidated the overall properties of these polymerases, including how they interact with other components of the replisome, but have only begun to define the enzymatic mechanism of nucleotide incorporation. Using transient-state methods, we have determined the kinetic mechanism of accurate replication by PolC, the replicative polymerase from the Gram-positive pathogen Staphylococcus aureus. Remarkably, PolC can recognize the presence of the next correct nucleotide prior to completing the addition of the current nucleotide. By modulating the rate of pyrophosphate byproduct release, PolC can tune the speed of DNA synthesis in response to the concentration of the next incoming nucleotide. The kinetic mechanism described here would allow PolC to perform high fidelity replication in response to diverse cellular environments.     

Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production

Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both gram-negative and -positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP) and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to date. In order to study this regulation further, we performed a screen to find inhibitors of the phosphatase, CpsB. This led to the observation that a recently discovered marine sponge metabolite, fascioquinol E, inhibited CpsB phosphatase activity both in vitro and in vivo at concentrations that did not affect the growth of the bacteria. This inhibition resulted in decreased capsule synthesis in D39 and Type 1 S. pneumoniae. Furthermore, concentrations of Fascioquinol E that inhibited capsule also lead to increased attachment of pneumococci to a macrophage cell line, suggesting that this compound would inhibit the virulence of the pathogen. Interestingly, this compound also inhibited the phosphatase activity of the structurally unrelated gram-negative PTP, Wzb, which belongs to separate family of protein tyrosine phosphatases. Furthermore, incubation with Klebsiella pneumoniae, which contains a homologous phosphatase, resulted in decreased capsule synthesis. Taken together, these data provide evidence that PTPs are critical for Wzy-dependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.     

Cross-utilization of the beta sliding clamp by replicative polymerases of evolutionary divergent organisms

Chromosomal replicases are multiprotein machines comprised of a DNA polymerase, a sliding clamp, and a clamp loader. This study examines replicase components for their ability to be switched between Gram-positive and Gram-negative organisms. These two cell types diverged over 1 billion years ago, and their sequences have diverged widely. Yet the Escherichia coli beta clamp binds directly to Staphylococcus aureus PolC and makes it highly processive, confirming and extending earlier results (Low, R. L., Rashbaum, S. A. , and Cozzarelli, N. R. (1976) J. Biol. Chem. 251, 1311-1325). We have also examined the S. aureus beta clamp. The results show that it functions with S. aureus PolC, but not with E. coli polymerase III core. PolC is a rather potent polymerase by itself and can extend a primer with an intrinsic speed of 80-120 nucleotides per s. Both E. coli beta and S. aureus beta converted PolC to a highly processive polymerase, but surprisingly, beta also increased the intrinsic rate of DNA synthesis to 240-580 nucleotides per s. This finding expands the scope of beta function beyond a simple mechanical tether for processivity to include that of an effector that increases the intrinsic rate of nucleotide incorporation by the polymerase.     

CpsB is a modulator of capsule-associated tyrosine kinase activity in Streptococcus pneumoniae

Tyrosine phosphorylation is associated with polysaccharide synthesis in a number of Gram-positive and Gram-negative bacteria. In Streptococcus pneumoniae, CpsB, CpsC, and CpsD affect tyrosine phosphorylation and are critical for the production of a mature capsule in vitro. To characterize the interactions between these proteins and the phosphorylation event they modulate, cps2B, cps2C, and cps2D from the capsule type 2 S. pneumoniae D39 were cloned and expressed both individually and in combination in Escherichia coli. Cps2D purified from E. coli was not phosphorylated unless it was co-expressed with its cognate transmembrane domain, Cps2C. Purified phosphorylated Cps2D had tyrosine kinase activity and could phosphorylate both dephosphorylated Cps2D and an exogenous substrate (poly-Glu-Tyr) in the absence of ATP. Cps2B exhibited phosphatase activity against both purified phosphorylated Cps2D and p-nitrophenyl phosphate. An additional role for Cps2B as an inhibitor of Cps2D phosphorylation was demonstrated in both co-expression experiments in E. coli and in vitro experiments where it blocked the transphosphorylation of Cps2D even in the presence of the phosphatase inhibitor sodium orthovanadate. cps2C and cps2D deletion mutants in S. pneumoniae produced no detectable mature capsule during laboratory culture. Both were avirulent in systemic mouse infections and were unable to colonize the nasopharynx, suggesting that the failure to produce capsule was not dependent on the environment. Based on these results, we propose a model for capsule regulation where CpsB, CpsC, CpsD, and ATP form a stable complex that enhances capsule synthesis.     

The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair

Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3′-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3′-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3′-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3′-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER.     

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.