Palm Subdomains Research Articles

Structure of Foot-and-Mouth Disease Virus RNA-dependent RNA Polymerase and Its Complex with a Template-Primer RNA

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.

Structure-specific DNA-induced Conformational Changes in Taq Polymerase Revealed by Small Angle Neutron Scattering

The DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase domain for synthesizing a new DNA strand and a 5'-nuclease domain for cleaving RNA primers or damaged DNA strands. The extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that occur in Taq polymerase upon binding "overlap flap" DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope from small angle neutron scattering data shows that in the DNA-bound form, the nuclease domain is lifted up relative to its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer of the DNA substrate from one active site to the other.

Systematic Investigation of Non-Nucleoside Inhibitors of HIV-1 Reverse Transcriptase (NNRTIs)

The 44 crystal structures of NNRTIs complexed with HIV-1 reverse transcriptase (RT) have been analyzed in detail especially the orientation geometries and the distances between inhibitor molecules and surrounding amino acids. In general, various NNIs bind to the same region of the HIV-1 RT in the palm subdomain of p66, but subtle differences in individual interactions between RT and its inhibitors can be detected by comparison of all structures in this study.

A positively charged side chain at position 154 on the beta8-alphaE loop of HIV-1 RT is required for stable ternary complex formation.

Lys154 is the only positively charged residue located in the VLPQGWK motif on the beta8-alphaE loop at the junction of the fingers and palm subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Some of the conserved residues in this motif are critical for RT function, while others have been shown to confer nucleoside drug resistance and fidelity to the enzyme. In order to understand the functional implication of this positively charged residue, we carried out site-directed mutagenesis at position 154 and biochemically characterized the mutant enzymes. Mutants carrying negatively charged side chains (K154D and K154E) were severely impaired in their polymerase function, while those with hydrophobic side chains (K154A and K154I) were moderately affected. Analysis of the binary complexes formed by these mutants revealed that all the mutant derivatives retained their ability to form an enzyme template primer (E-TP) binary complex similar to the wild-type enzyme. In contrast, their ability to form stable E-TP-dNTP ternary complexes varied greatly and was dependent on the nature of the side chain at position 154. The conservative Lys-->Arg mutant was not affected in its ability to form a stable ternary complex, while those carrying non-polar or negatively charged side chains were significantly impaired. The apparent K(d [dNTP]) values for these non-conservative mutants were approximately 16- to 400-fold higher than the wild-type enzyme, indicating that a positively charged side chain at position 154 may be required for efficient formation of a stable ternary complex. Interestingly, all the mutant derivatives of Lys154 were completely resistant to a nucleoside analog inhibitor, 3'-dideoxy 3'-thiacytidine (3TC), implying that Lys154 may play a role in conferring 3TC sensitivity to HIV-1 RT. These findings are discussed in the context of the binary and ternary complex crystal structures of HIV-1 RT.

Human immunodeficiency virus type 2 reverse transcriptase activity in model systems that mimic steps in reverse transcription.

Human immunodeficiency virus type 2 (HIV-2) infection is a serious problem in West Africa and Asia. However, there have been relatively few studies of HIV-2 reverse transcriptase (RT), a potential target for antiviral therapy. Detailed knowledge of HIV-2 RT activities is critical for development of specific high-throughput screening assays of potential inhibitors. Here, we have conducted a systematic evaluation of HIV-2 RT function, using assays that model specific steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes were comparable. However, when the RT concentration was significantly reduced, HIV-2 RT was less active than the HIV-1 enzyme. HIV-2 RT was also impaired in its ability to catalyze secondary RNase H cleavage in assays that mimic tRNA primer removal during plus-strand transfer and degradation of genomic RNA fragments during minus-strand DNA synthesis. In addition, initiation of plus-strand DNA synthesis was much less efficient with HIV-2 RT than with HIV-1 RT. This may reflect architectural differences in the primer grip regions in the p66 (HIV-1) and p68 (HIV-2) palm subdomains of the two enzymes. The implications of our findings for antiviral therapy are discussed.

Mutagenesis of Cysteine 280 of the Reverse Transcriptase of Human Immunodeficiency Virus Type-1: The Effects on the Ribonuclease H Activity

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RT of human immunodeficiency virus type-1 (HIV-1) is composed of two subunits. The p51, which is the smaller subunit, shares with the larger p66 subunit the same amino-terminal part (which encompasses the DNA polymerase domain) and lacks the carboxyl-terminal segment of the p66 (which is the RNase H domain). The structure of the polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains) linked to the RNase H domain. Chemical modifications by thiol-specific reagents of cysteine 280, located in α helix I in the thumb subdomain of the polymerase domain, affect substantially only the RNase H activity. Also, the substitution of a serine for C280 did not alter any of the RT activities. Here we have systematically modified the C280 residue to either of the following residues: W, P, H, L, M, Y, Q, E or R. Only the first two mutations lead to a marked reduction in the RNase H activity, whereas none of the mutations affected the polymerase function to a significant extent. As expected, due to their impaired RNase H, the C280W and C280P mutants also had a very low DNA strand-transfer activity. It is also apparent from subunit-directed mutagenesis that each of the RT subunits contributes to the level of RNase H activity, yet the contribution of the p51 subunit to this activity is somewhat higher than that of the p66. Steady-state kinetic analyses have indicated that the RNase H activity was reduced mainly due to the sharp increase in the K m rather than changes in the k cat values. This suggests that the modifications of C280 lead to an impaired affinity of HIV-1 RT towards the RNA–DNA substrate.

Substitution of conserved hydrophobic residues in motifs B and C of HIV-1 RT alters the geometry of its catalytic pocket.

Recent crystallographic data suggest that a number of hydrophobic residues seen clustered between the structurally conserved alphabetabetaalpha motif of the palm subdomain and at the junction of palm and fingers subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) provide an optimal geometry to the alphabeta sandwich of the palm subdomain, which harbors the catalytic site and the primer-binding grip region. This region has also been implicated in binding to the non-nucleoside RT inhibitors. We have evaluated the impact of conserved and nonconserved amino acid substitutions at four hydrophobic positions in this region of HIV-1 RT, in the context of their biochemical characteristics. The residues that have been analyzed include Ile-167, Leu-187, and Val-189 which are located within the alphabetabetaalpha motif, while Trp-153 lies next to the conserved LPQG motif, at the juncture of the palm and fingers subdomains. Our results show that all substitutions at I167 with the exception of I167T were deleterious to enzyme function in contrast to substitutions at V189 which enhanced the enzymatic activity. Ala substitution at residues W153 and L187 also substantially hindered the polymerase function of the enzyme. Further analysis revealed that the defective mutant derivatives of I167 were substantially impaired in their apparent dNTP binding abilities, thereby impacting the geometry of the dNTP binding pocket. The extent of misinsertion and misincorporation was higher in the case of RT variants of W153 and V189, specifically on a DNA template. Interestingly, none of the mutant derivatives of these residues were resistant to nucleoside inhibitors. A salient finding was that all nonconserved mutants of these residues exhibited hypersensitivity to nevirapine. We have analyzed these findings and their significance in the context of the HIV-1 RT structure and propose that these residues exert their effect via their indirect interactions with the template-primer through residues in their vicinity.

The palm subdomain-based active site is internally permuted in viral RNA-dependent RNA polymerases of an ancient lineage.

Template-dependent polynucleotide synthesis is catalyzed by enzymes whose core component includes a ubiquitous αβ palm subdomain comprising A, B and C sequence motifs crucial for catalysis. Due to its unique, universal conservation in all RNA viruses, the palm subdomain of RNA-dependent RNA polymerases (RdRps) is widely used for evolutionary and taxonomic inferences. We report here the results of elaborated computer-assisted analysis of newly sequenced replicases from Thosea asigna virus (TaV) and the closely related Euprosterna elaeasa virus (EeV), insect-specific ssRNA+ viruses, which revise a capsid-based classification of these viruses with tetraviruses, an Alphavirus-like family. The replicases of TaV and EeV do not have characteristic methyltransferase and helicase domains, and include a putative RdRp with a unique C–A–B motif arrangement in the palm subdomain that is also found in two dsRNA birnaviruses. This circular motif rearrangement is a result of migration of ∼22 amino acid (aa) residues encompassing motif C between two internal positions, separated by ∼110 aa, in a conserved region of ∼550 aa. Protein modeling shows that the canonical palm subdomain architecture of poliovirus (ssRNA+) RdRp could accommodate the identified sequence permutation through changes in backbone connectivity of the major structural elements in three loop regions underlying the active site. This permutation transforms the ferredoxin-like β1αAβ2β3αBβ4 fold of the palm subdomain into the β2β3β1αAαBβ4 structure and brings β-strands carrying two principal catalytic Asp residues into sequential proximity such that unique structural properties and, ultimately, unique functionality of the permuted RdRps may result. The permuted enzymes show unprecedented interclass sequence conservation between RdRps of true ssRNA+ and dsRNA viruses and form a minor, deeply separated cluster in the RdRp tree, implying that other, as yet unidentified, viruses may employ this type of RdRp. The structural diversification of the palm subdomain might be a major event in the evolution of template-dependent polynucleotide polymerases in the RNA–protein world.

The Mutational Specificity of the Dbh Lesion Bypass Polymerase and Its Implications

The Dbh polymerase of Sulfolobus solfataricus is a member of the recently described family of low fidelity DNA polymerases involved in bypass of DNA lesions. To investigate the enzymatic properties of Dbh, we characterized the errors made by this polymerase in vitro. Not only is Dbh much less accurate than the "classical" polymerases, but it showed a remarkable tendency to skip over a template pyrimidine positioned immediately 3' to a G residue, generating a single-base deletion. Single-turnover kinetic measurements suggest possible mechanisms. First, Dbh shows a bias in favor of dCTP, such that the rate of incorporation of dCTP opposite a template G is about 10-fold faster than for the other three dNTPs opposite their complementary partners. On a DNA substrate corresponding to a frameshift hotspot, the rate of frameshift insertion of dCTP opposite a template G that is one residue 5' to the expected templating position is approximately equal to the rate of the non-frameshifted C-dGTP insertion. We suspect that the unusual mutational specificity of Dbh (which is shared with other polymerases from the DinB branch of the bypass polymerase family) may be related to the type of DNA lesion(s) that it serves to bypass in vivo.

Structure-Function Relationships of the RNA-dependent RNA Polymerase from Poliovirus (3Dpol): A SURFACE OF THE PRIMARY OLIGOMERIZATION DOMAIN FUNCTIONS IN CAPSID PRECURSOR PROCESSING AND VPg URIDYLYLATION

The primary oligomerization domain of poliovirus polymerase, 3Dpol, is stabilized by the interaction of the back of the thumb subdomain of one molecule with the back of the palm subdomain of a second molecule, thus permitting the head-to-tail assembly of 3Dpol monomers into long fibers. The interaction of Arg-455 and Arg-456 of the thumb with Asp-339, Ser-341, and Asp-349 of the palm is key to the stability of this interface. We show that mutations predicted to completely disrupt this interface do not produce equivalent growth phenotypes. Virus encoding a polymerase with changes of both residues of the thumb to alanine is not viable; however, virus encoding a polymerase with changes of all three residues of the palm to alanine is viable. Biochemical analysis of 3Dpol derivatives containing the thumb or palm substitutions revealed that these derivatives are both incapable of forming long fibers, suggesting that polymerase fibers are not essential for virus viability. The RNA binding activity, polymerase activity, and thermal stability of these derivatives were equivalent to that of the wild-type enzyme. The two significant differences observed for the thumb mutant were a modest reduction in the ability of the altered 3CD proteinase to process the VP0/VP3 capsid precursor and a substantial reduction in the ability of the altered 3Dpol to catalyze oriI-templated uridylylation of VPg. The defect to uridylylation was a result of the inability of 3CD to stimulate this reaction. Because 3C alone can substitute for 3CD in this reaction, we conclude that the lethal replication phenotype associated with the thumb mutant is caused, in part, by the disruption of an interaction between the back of the thumb of 3Dpol and some undefined domain of 3C. We speculate that this interaction may also be critical for assembly of other complexes required for poliovirus genome replication.

Mechanism of Human Telomerase Inhibition by BIBR1532, a Synthetic, Non-nucleosidic Drug Candidate

Telomerase, a ribonucleoprotein acting as a reverse transcriptase, has been identified as a target for cancer drug discovery. The synthetic, non-nucleosidic compound, BIBR1532, is a potent and selective telomerase inhibitor capable of inducing senescence in human cancer cells (). In the present study, the mode of drug action was characterized. BIBR1532 inhibits the native and recombinant human telomerase, comprising the human telomerase reverse transcriptase and human telomerase RNA components, with similar potency primarily by interfering with the processivity of the enzyme. Enzyme-kinetic experiments show that BIBR1532 is a mixed-type non-competitive inhibitor and suggest a drug binding site distinct from the sites for deoxyribonucleotides and the DNA primer, respectively. Thus, BIBR1532 defines a novel class of telomerase inhibitor with mechanistic similarities to non-nucleosidic inhibitors of HIV1 reverse transcriptase.

PII: S0969-2126(01)00700-6

PII: S0969-2126(01)00700-6

The reverse transcriptase sequence of human immunodeficiency virus type 1 is under positive evolutionary selection within the central nervous system.

The human immunodeficiency virus type 1 (HIV-1) enters the central nervous system (CNS) during the acute phase of infection and causes AIDS-related encephalitis and dementia in 30% of individuals. Previous studies show that HIV-1 sequences derived from the CNS of infected patients, including the sequence encoding reverse transcriptase (RT), are genetically distinct from sequences in other tissues. The hypothesis of the current study is that the RT sequence of HIV-1 is under positive selection within the CNS. Multiple alignments of non-CNS-derived and CNS-derived HIV-1 RT sequences were constructed using the ClustalW 1.8 program. The multiple alignments were analyzed with the Synonymous/Nonsynonymous Analysis Program. Codon positions 122-125, 135-149, and 166-212 of the CNS-derived RT sequences underwent a greater accumulation of nonsynonymous than synonymous substitutions, which was markedly different from the analysis results of the non-CNS-derived RT sequences. These residues are located in the finger and palm subdomains of the RT protein structure, which encodes the polymerase active site. The analysis of CNS-derived partial-length RT sequences that encompass these regions yielded similar results. A comparison of CNS-derived RT sequences to a non-CNS-derived RT consensus sequence revealed that a majority of the nonsynonymous substitutions resulted in a specific amino acid replacement. These results indicate that reverse transcriptase is under positive selection within the CNS. The amino acid replacements were visualized on a three-dimensional structure of HIV-1 RT using the Sybyl software suite. The protein structure analysis revealed that the amino acid replacements observed among the CNS-derived sequences occurred in areas of known structural and functional significance.

Functional Properties of a Monoclonal Antibody Inhibiting the Hepatitis C Virus RNA-dependent RNA Polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), has recently emerged as a promising target for antiviral intervention. Here, we describe the isolation, functional characterization, and molecular cloning of a monoclonal antibody (mAb) inhibiting the HCV RdRp. This mAb, designated 5B-12B7, binds with high affinity to a conformational epitope in the palm subdomain of the HCV RdRp and recognizes native NS5B expressed in the context of the entire HCV polyprotein or subgenomic replicons. Complete inhibition of RdRp activity in vitro was observed at equimolar concentrations of NS5B and mAb 5B-12B7, whereas RdRp activities of classical swine fever virus NS5B and poliovirus 3D polymerase were not affected. mAb 5B-12B7 selectively inhibited NTP binding to HCV NS5B, whereas binding of template RNA was unaffected, thus explaining the mechanism of action at the molecular level. The mAb 5B-12B7 heavy and light chain variable domains were cloned by reverse transcription-PCR, and a single chain Fv fragment was assembled for expression in Escherichia coli and in eukaryotic cells. The mAb 5B-12B7 single chain Fv fragment bound to NS5B both in vitro and in transfected human cell lines and therefore may be potentially useful for intracellular immunization against HCV. More important, detailed knowledge of the mAb 5B-12B7 contact sites on the enzyme may facilitate the development of small molecule RdRp inhibitors as novel antiviral agents.

Solution structure of a viral DNA repair polymerase.

DNA polymerase X (Pol X) from the African swine fever virus (ASFV) specifically binds intermediates in the single-nucleotide base-excision repair process, an activity indicative of repair function. In addition, Pol X catalyzes DNA polymerization with low nucleotide-insertion fidelity. The structural mechanisms by which DNA polymerases confer high or low fidelity in DNA polymerization remain to be elucidated. The three-dimensional structure of Pol X has been determined. Unlike other DNA polymerases, Pol X is formed from only a palm and a C-terminal subdomain. Pol X has a novel palm subdomain fold, containing a positively charged helix at the DNA binding surface. Purine deoxynucleoside triphosphate (dNTP) substrates bind between the palm and C-terminal subdomain, at a dNTP-binding helix, and induce a unique conformation in Pol X. The purine dNTP-bound conformation and high binding affinity for dGTP-Mg(2+) of Pol X may contribute to its low fidelity.

Solution structure of a viral DNA polymerase X and evidence for a mutagenic function.

The African swine fever virus DNA polymerase X (ASFV Pol X or Pol X), the smallest known nucleotide polymerase, has recently been reported to be an extremely low fidelity polymerase that may be involved in strategic mutagenesis of the viral genome. Here we report the solution structure of Pol X. The structure, unique within the realm of nucleotide polymerases, consists of only palm and fingers subdomains. Despite the absence of a thumb subdomain, which is important for DNA binding in other polymerases, we show that Pol X binds DNA with very high affinity. Further structural analyses suggest a novel mode of DNA binding that may contribute to low fidelity synthesis. We also demonstrate that the ASFV DNA ligase is a low fidelity ligase capable of sealing a nick that contains a G-G mismatch. This supports the hypothesis of a virus-encoded, mutagenic base excision repair pathway consisting of a tandem Pol X/ligase mutator.

The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is affected by the thumb subdomain of the small protein subunits

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RTs of HIV-1 and HIV-2 are heterodimers of p66/p51 and p68/p54 subunits, respectively. The smaller subunit lacks the C-terminal segment of the larger subunit (which is the RNase H domain). The structure of the DNA polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains), linked to the RNase H domain via the connection subdomain. The RNase H activity of the Rod strain of HIV-2 RT is about tenfold lower than that of HIV-1 RT, while the DNA polymerase activity of these RTs is similar. A chimeric RT in which residues 227–427 (which constitute a small part of the palm and the entire thumb and connection subdomains) of the Rod strain of HIV-2 RT were replaced by the corresponding segment from HIV-1 RT, has an RNase H activity as high as HIV-1 RT (despite the fact that the RNase H domain is derived from HIV-2 RT).We analyzed the RNase H activity of wild-type HIV-2 RT from the D-194 strain and compared it with this activity of the RT from the Rod strain of HIV-2 and HIV-1 RT. The level of this activity of both HIV-2 RT strains was low; suggesting that low RNase H activity is a general property of HIV-2 isolates. The in vitro RNase H digestion pattern of the three wild-type RTs was indistinguishable, despite the difference in the level of RNase H activity. We constructed new chimeric HIV-1/HIV-2 RTs, in which protein segments and/or subunits were exchanged. The DNA polymerase activity of the parental HIV-1 and HIV-2 RTs was similar; as expected, the specific activity of the polymerases of all the hybrid RTs were also similar. However, the RNase H specific activity of the chimeric RTs was either high (like HIV-1 RT) or low (like HIV-2 RT). The origin of the thumb subdomain in the small subunit of the chimeric RTs (residues 244–322) determines the level of the RNase H activity. The strand-transfer activity of the chimeric RTs is also affected by the thumb subdomain of the small subunit; transfer was much more efficient if this subdomain was derived from HIV-1 RT. The data can be explained from the three-dimensional structure of HIV-1 RT. The thumb of the smaller subunit contacts the RNase H domain; it is through these contacts that the thumb affects the level of the RNase H activity of RT.

Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus nonstructural protein 5B polymerase

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A 9C 9, 16D 9A 4 and 20A 12C 7, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A 9C 9, and between 282 and 372 for 16D 9A 4 and 20A 12C 7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.

Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

1H, 15N and 13C resonance assignments for the gallium protoporphyrin IX-HasA(sm) hemophore complex.

The African Swine Fever Virus (ASFV) is known to cause a lethal disease in domestic pigs. The virus contains two DNA polymerases; a eukaryotic-like DNA polymerase involved in viral DNA replication and a 174 amino acid DNA polymerase that is a member of the polymerase X family (Pol X). This family includes DNA polymerase β (β-Pol), terminal transferase (TdT), DNA polymerase μ (Pol μ) (Dominguez et al., 2000), and DNA polymeraseλ (Pol λ) (Aoufouchi et al., 2000). One notable difference between Pol X and the Pol X family is the absence of N-terminal DNA binding domains that are found in β-Pol, TdT, Pol μ, and Pol λ and the absence of BRCT domains that are found in Pol μ and Pol λ. In comparison to β-Pol, Pol X comprises only the palm and C-terminal subdomains. Based on biochemical analysis, Pol X was shown to be similar to β-Pol with respect to template-directed DNA synthesis, preference for deoxynucleotides versus ribonucleotides, and repair of a single nucleotide gap DNA (Oliveros et al., 1997). This would suggest that Pol X acts in base excision repair of the viral DNA inside the cytoplasm of the host cell. In addition, Pol X catalyzes the formation of a G:G mismatch with relatively high catalytic efficiency (Showalter and Tsai, 2001) and may provide mutase function to ASFV. As the smallest DNA polymerase, Pol X represents the minimal core needed for a protein to act as a template directed nucleotidal transferase. Here we report on the nearly complete backbone and side chain 1H, 15N, and 13C resonance assignments for the 20 kDa Pol X.