Biomedical subjects

Lars C Pedersen

Publications and source records attributed to Lars C Pedersen.

At least 19 recordsLinked to original sources

PubMed2007

Modifying the beta,gamma leaving-group bridging oxygen alters nucleotide incorporation efficiency, fidelity, and the catalytic mechanism of DNA polymerase beta.

DNA polymerase catalysis and fidelity studies typically compare incorporation of "right" versus "wrong" nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the beta,gamma-bridging O with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) beta. T.G mismatches occur with fidelities similar to dGTP with the exception of the CH2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with Kds between 1 and 4 microM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brnsted correlations of log(kpol) versus leaving-group pKa for both right and wrong base incorporation reveal similar sensitivities (betalg approximately -0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pKas of CF2, O, and the sterically bulky CCl2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3'-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.

Base Sequence↗

PubMed2006

Structural insight into the substrate specificity of DNA Polymerase mu.

DNA polymerase mu (Pol mu) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol mu's unusual substrate specificity, we describe the 2.4 A crystal structure of the polymerase domain of murine Pol mu bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol mu that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol mu from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3' ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases.

Amino Acid Sequence↗

PubMed2006

The nuclease a-inhibitor complex is characterized by a novel metal ion bridge.

Nonspecific, extracellular nucleases have received enhanced attention recently as a consequence of the critical role that these enzymes can play in infectivity by overcoming the host neutrophil defense system. The activity of the cyanobacterial nuclease NucA, a member of the betabetaalpha Me superfamily, is controlled by the specific nuclease inhibitor, NuiA. Here we report the 2.3-A resolution crystal structure of the NucA-NuiA complex, showing that NucA inhibition by NuiA involves an unusual divalent metal ion bridge that connects the nuclease with its inhibitor. The C-terminal Thr-135(NuiA) hydroxyl oxygen is directly coordinated with the catalytic Mg(2+) of the nuclease active site, and Glu-24(NuiA) also extends into the active site, mimicking the charge of a scissile phosphate. NuiA residues Asp-75 and Trp-76 form a second interaction site, contributing to the strength and specificity of the interaction. The crystallographically defined interface is shown to be consistent with results of studies using site-directed NuiA mutants. This mode of inhibition differs dramatically from the exosite mechanism of inhibition seen with the DNase colicins E7/E9 and from other nuclease-inhibitor complexes that have been studied. The structure of this complex provides valuable insights for the development of inhibitors for related nonspecific nucleases that share the DRGH active site motif such as the Streptococcus pneumoniae nuclease EndA, which mediates infectivity of this pathogen, and mitochondrial EndoG, which is involved in recombination and apoptosis.

Amino Acid Motifs↗

PubMed2006

Anticoagulant heparan sulfate: structural specificity and biosynthesis.

Heparan sulfate (HS) is present on the surface of endothelial and surrounding tissues in large quantities. It plays important roles in regulating numerous functions of the blood vessel wall, including blood coagulation, inflammation response, and cell differentiation. HS is a highly sulfated polysaccharide containing glucosamine and glucuronic/iduronic acid repeating disaccharide units. The unique sulfated saccharide sequences of HS determine its specific functions. Heparin, an analog of HS, is the most commonly used anticoagulant drug. Because of its wide range of biological functions, HS has become an interesting molecule to biochemists, medicinal chemists, and developmental biologists. In this review, we summarize recent progress toward understanding the interaction between HS and blood-coagulating factors, the biosynthesis of anticoagulant HS and the mechanism of action of HS biosynthetic enzymes. Furthermore, knowledge of the biosynthesis of HS facilitates the development of novel enzymatic approaches to synthesize HS from bacterial capsular polysaccharides and to produce polysaccharide end products with high specificity for the biological target. These advancements provide the foundation for the development of polysaccharide-based therapeutic agents.

Anticoagulants↗

PubMed2006

Structure of DNA polymerase beta with a benzo[c]phenanthrene diol epoxide-adducted template exhibits mutagenic features.

We have determined the crystal structure of the human base excision repair enzyme DNA polymerase beta (Pol beta) in complex with a 1-nt gapped DNA substrate containing a template N2-guanine adduct of the tumorigenic (-)-benzo[c]phenanthrene 4R,3S-diol 2S,1R-epoxide in the gap. Nucleotide insertion opposite this adduct favors incorrect purine nucleotides over the correct dCMP and hence can be mutagenic. The structure reveals that the phenanthrene ring system is stacked with the base pair immediately 3' to the modified guanine, thereby occluding the normal binding site for the correct incoming nucleoside triphosphate. The modified guanine base is displaced downstream and prevents the polymerase from achieving the catalytically competent closed conformation. The incoming nucleotide binding pocket is distorted, and the adducted deoxyguanosine is in a syn conformation, exposing its Hoogsteen edge, which can hydrogen-bond with dATP or dGTP. In a reconstituted base excision repair system, repair of a deaminated cytosine (i.e., uracil) opposite the adducted guanine was dramatically decreased at the Pol beta insertion step, but not blocked. The efficiency of gap-filling dCMP insertion opposite the adduct was diminished by >6 orders of magnitude compared with an unadducted templating guanine. In contrast, significant misinsertion of purine nucleotides (but not dTMP) opposite the adducted guanine was observed. Pol beta also misinserts a purine nucleotide opposite the adduct with ungapped DNA and exhibits limited bypass DNA synthesis. These results indicate that Pol beta-dependent base excision repair of uracil opposite, or replication through, this bulky DNA adduct can be mutagenic.

DNA Adducts↗

PubMed2006

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex.

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.

Amino Acid Sequence↗

PubMed2006

Energy analysis of chemistry for correct insertion by DNA polymerase beta.

X-ray crystallographic structures of human DNA polymerase beta with nonhydrolyzable analogs containing all atoms in the active site required for catalysis provide a secure starting point for a theoretical analysis (quantum mechanics/molecular mechanics) of the mechanism of chemistry without biasing of modeling assumptions as required in previous studies. These structures provide the basis for a detailed quantum mechanics/molecular mechanics study of the path for the complete transfer of a monophosphate nucleoside donor to the sugar acceptor in the active site. The reaction is largely associative with the main energetic step preceded by proton transfer from the terminal primer deoxyribose O3' to Asp-256. The key residues that provide electrostatic stabilization of the transition state are identified and compared with those identified by mutational studies.

Binding Sites↗

PubMed2006

Promiscuous mismatch extension by human DNA polymerase lambda.

DNA polymerase lambda (Pol lambda) is one of several DNA polymerases suggested to participate in base excision repair (BER), in repair of broken DNA ends and in translesion synthesis. It has been proposed that the nature of the DNA intermediates partly determines which polymerase is used for a particular repair reaction. To test this hypothesis, here we examine the ability of human Pol lambda to extend mismatched primer-termini, either on 'open' template-primer substrates, or on its preferred substrate, a 1 nt gapped-DNA molecule having a 5'-phosphate. Interestingly, Pol lambda extended mismatches with an average efficiency of approximately 10(-2) relative to matched base pairs. The match and mismatch extension catalytic efficiencies obtained on gapped molecules were approximately 260-fold higher than on template-primer molecules. A crystal structure of Pol lambda in complex with a single-nucleotide gap containing a dG.dGMP mismatch at the primer-terminus (2.40 A) suggests that, at least for certain mispairs, Pol lambda is unable to differentiate between matched and mismatched termini during the DNA binding step, thus accounting for the relatively high efficiency of mismatch extension. This property of Pol lambda suggests a potential role as a 'mismatch extender' during non-homologous end joining (NHEJ), and possibly during translesion synthesis.

Base Pair Mismatch↗

PubMed2006

Structural analysis of strand misalignment during DNA synthesis by a human DNA polymerase.

Insertions and deletions in coding sequences can alter the reading frame of genes and have profound biological consequences. In 1966, Streisinger proposed that these mutations result from strand slippage, which in repetitive sequences generates misaligned intermediates stabilized by correct base pairing that support polymerization. We report here crystal structures of human DNA polymerase lambda, which frequently generates deletion mutations, bound to such intermediates. Each contains an extrahelical template nucleotide upstream of the active site. Surprisingly, the extra nucleotide, even when combined with an adjacent mismatch, does not perturb polymerase active site geometry, which is indistinguishable from that for correctly aligned strands. These structures reveal how pol lambda can polymerize on substrates with minimal homology during repair of double-strand breaks and represent strand-slippage intermediates consistent with Streisinger's classical hypothesis. They are thus relevant to the origin of single-base deletions, a class of mutations that can confer strong biological phenotypes.

Binding Sites↗

PubMed2006

Magnesium-induced assembly of a complete DNA polymerase catalytic complex.

The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3'-OH and catalytic Mg2+, resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here, we present a crystal structure of a precatalytic complex of a DNA polymerase with bound substrates that include the primer 3'-OH and catalytic Mg2+. This catalytic intermediate was trapped with a nonhydrolyzable deoxynucleotide analog. Comparison with two new structures of DNA polymerase beta lacking the 3'-OH or catalytic Mg2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3' on the alphaP of the incoming nucleotide.

Binding Sites↗

PubMed2005

Enzymatic redesigning of biologically active heparan sulfate.

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3'-phosphoadenosine 5'-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.

Animals↗

PubMed2005

Structure-function studies of DNA polymerase lambda.

DNA polymerase lambda is a member of the X family of polymerases that is implicated in non-homologous end-joining of double-strand breaks in DNA and in base excision repair of DNA damage. To better understand the roles of DNA polymerase lambda in these repair pathways, here we review its structure and biochemical properties, with emphasis on its gap-filling polymerization activity, its dRP lyase activity and its unusual DNA synthetic (in)fidelity.

DNA Polymerase beta↗

PubMed2005

Structural insight into the DNA polymerase beta deoxyribose phosphate lyase mechanism.

A large number of biochemical and genetic studies have demonstrated the involvement of DNA polymerase beta (Pol beta) in mammalian base excision repair (BER). Pol beta participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5'-deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol beta complexed with 5'-phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1' of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120 degrees about the dRP 3'-phosphate is required to position the epsilon-amino Lys72 close to the dRP C1'. This model also suggests that several other side chains are in position to facilitate the beta-elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity.

Binding Sites↗

PubMed2005

Thr176 regulates the activity of the mouse nuclear receptor CAR and is conserved in the NR1I subfamily members PXR and VDR.

The mouse nuclear receptor CAR (constitutively active receptor) is a transcription factor that is activated by phenobarbital-type inducers such as TCPOBOP {1,4 bis[2-(3,5-dichloropyridyloxy)]benzene} in liver in vivo. However, CAR is constitutively active in cell-based transfection assays, the molecular mechanism for which has not been elucidated yet. In the model structure of CAR, Thr176 constitutes a part of the ligand-binding surface, but its side chain is not directed toward the surface, instead it forms a hydrogen bond with Thr350 in the AF2 (activation function 2) domain of CAR. Thr350 is known to regulate CAR activity [Ueda, Kakizaki, Negishi, and Sueyoshi (2002) Mol. Pharmacol. 61, 1284-1288]. Thr176 was mutated to various amino acids to examine whether this interaction played a role in conferring the constitutive activity. Hydrophobic and positively charged amino acids at position 176 abrogated the constitutive activity, whereas polar and negatively charged amino acids retained it. When one of the small hydrophobic amino acids, such as alanine or valine, was substituted for threonine, the mutants were fully activated by TCPOBOP. The co-activator SRC-1 (steroid receptor co-activator-1) regulated the activity changes associated with the mutations. Thr248 and Ser230 are the Thr176-corresponding residues in human pregnane X receptor and mouse vitamin D3 receptor respectively, interacting directly with the conserved threonine in the AF2 domains. Thr248 and Ser230 also regulated the ligand-dependent activity of these receptors by augmenting binding of the receptors to SRC-1. Thr176, Thr248 and Ser230 are conserved residues in the NR1I (nuclear receptor 1I) subfamily members and determine their activity.

Amino Acid Sequence↗

PubMed2005

Structural insights into the mechanism of nuclease A, a betabeta alpha metal nuclease from Anabaena.

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and double-stranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the delta(2-24),D121A mutant of NucA in the presence of Zn2+ and Mn2+ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a betabeta alpha metal finger motif and a hydrated Mn2+ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites approximately 26 A apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg57 in Serratia, the structure determined here indicates that Arg93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the betabeta alpha metal finger nuclease family and available mutational data on NucA, we propose that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state.

Amino Acid Motifs↗

PubMed2005

Nucleotide-induced DNA polymerase active site motions accommodating a mutagenic DNA intermediate.

DNA polymerases occasionally insert the wrong nucleotide. For this error to become a mutation, the mispair must be extended. We report a structure of DNA polymerase beta (pol beta) with a DNA mismatch at the boundary of the polymerase active site. The structure of this complex indicates that the templating adenine of the mispair stacks with the primer terminus adenine while the templating (coding) cytosine is flipped out of the DNA helix. Soaking the crystals of the binary complex with dGTP resulted in crystals of a ternary substrate complex. In this case, the templating cytosine is observed within the DNA helix and forms Watson-Crick hydrogen bonds with the incoming dGTP. The adenine at the primer terminus has rotated into a syn-conformation to interact with the opposite adenine in a planar configuration. Yet, the 3'-hydroxyl on the primer terminus is out of position for efficient nucleotide insertion.

Crystallography, X-Ray↗

PubMed2004

A closed conformation for the Pol lambda catalytic cycle.

Pol lambda is a family X member believed to fill short gaps during DNA repair. Here we report crystal structures of Pol lambda representing three steps in filling a single-nucleotide gap. These structures indicate that, unlike other DNA polymerases, Pol lambda does not undergo large subdomain movements during catalysis, and they provide a clear characterization of the geometry and stereochemistry of the in-line nucleotidyl transfer reaction.

Catalysis↗

PubMed2004

Structural analysis of the sulfotransferase (3-o-sulfotransferase isoform 3) involved in the biosynthesis of an entry receptor for herpes simplex virus 1.

Heparan sulfate (HS) plays essential roles in assisting herpes simplex virus infection and other biological processes. The biosynthesis of HS includes numerous specialized sulfotransferases that generate a variety of sulfated saccharide sequences, conferring the selectivity of biological functions of HS. We report a structural study of human HS 3-O-sulfotransferase isoform 3 (3-OST-3), a key sulfotransferase that transfers a sulfuryl group to a specific glucosamine in HS generating an entry receptor for herpes simplex virus 1. We have obtained the crystal structure of 3-OST-3 at 1.95 A in a ternary complex with 3'-phosphoadenosine 5'-phosphate and a tetrasaccharide substrate. Mutational analyses were also performed on the residues involved in the binding of the substrate. Residues Gln255 and Lys368 are essential for the sulfotransferase activity and lie within hydrogen bonding distances to the carboxyl and sulfo groups of the uronic acid unit. These residues participate in the substrate recognition of 3-OST-3. This structure provides atomic level evidence for delineating the substrate recognition and catalytic mechanism for 3-OST-3.

Amino Acid Sequence↗