William Konigsberg

William Konigsberg
Yale University | YU · Department of Molecular Biophysics and Biochemistry

PhD

About

310
Publications
14,194
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
15,198
Citations

Publications

Publications (310)
Article
Full-text available
Almost all DNA polymerases (pols) exhibit bell-shaped activity curves as a function of both pH and Mg2+ concentration. The pol activity is reduced when the pH deviates from the optimal value. When the pH is too low the concentration of a deprotonated general base (namely, the attacking 3'-hydroxyl of the 3' terminal residue of the primer strand) is...
Article
Full-text available
A minimal replication-transcription complex (RTC) of SARS-CoV-2 for synthesis of viral RNAs includes the nsp12 RNA-dependent RNA polymerase and two nsp8 RNA primase subunits for de novo primer synthesis, one nsp8 in complex with its accessory nsp7 subunit and the other without it. The RTC is responsible for faithfully copying the entire (+) sense v...
Article
Full-text available
Although Mg²⁺ is the metal ion that functions as the cofactor for DNA polymerases (DNA pols) in vivo, Mn²⁺ can also serve in this capacity but it reduces base discrimination. Metal ions aside from Mg²⁺ or Mn²⁺ can act as cofactors for some DNA pols but not for others. Here we report on the ability of several divalent metal ions to substitute for Mg...
Article
Full-text available
There is increasing interest in the use of DNA polymerases (DNA pols) in next-generation sequencing strategies. These methodologies typically rely on members of the A and B family of DNA polymerases that are classified as high-fidelity DNA polymerases. These enzymes possess the ability to selectively incorporate the correct nucleotide opposite a te...
Article
DNA polymerases (DNA pols) are essential for accurately copying genomes of all organisms. The polymerase and exonuclease activities associated with DNA pols require the presence of two divalent cations which occupy the A and B metal ion sites. Even though the two-metal ion mechanism is generally applicable for all DNA pols, a third metal ion was pr...
Article
Full-text available
Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two...
Article
Full-text available
Although Mg2+ is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn2+ can also serve but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg2+ or Mn2+ can also act as cofactors depending on the specific DNA po...
Article
Background: Individuals referred for stress testing to identify coronary artery disease may have nonobstructive atherosclerosis, which is not detected by stress tests. Identification of increased risk despite a negative stress test could inform prevention efforts. Abnormal ankle-brachial index (ABI) is associated with increased cardiovascular risk...
Article
Raman difference spectroscopy is used to monitor the conformational changes associated with dNMP incorporation in single crystals of a RB69 DNA polymerase (RB69pol) ternary complex. An inactive crystal of L415G RB69pol exo- with a primer/template duplex and a cognate dATP was produced in the presence of Ca2+. The Raman spectrum of the crystallized...
Article
This review will summarize our structural and kinetic studies on RB69 DNA polymerase (RB69pol) as well as on selected variants of the wild type enzyme that were undertaken to obtain a deeper understanding of the exquisitely high fidelity of B family replicative DNA polymerases. We discuss how the structures of the various RB69pol ternary complexes...
Article
Recent structures of DNA polymerase complexes with dGMPCPP/dT and dCTP/dA mispairs at the insertion site have shown that they adopt Watson-Crick geometry in the presence of Mn(2+) indicating that the tautomeric or ionization state of the base has changed. To see whether the tautomeric or ionization state of base-pair could be affect by its microenv...
Article
Full-text available
Internal cavities are a common feature of many proteins, often having profound effects on the dynamics of their interactions with substrate and binding partners. RB69 DNA polymerase (pol) has a hydrophobic cavity right below the nucleotide binding pocket at the tip of highly conserved L415 side chain. Replacement of this residue with Gly or Met in...
Article
Current hypotheses that attempt to rationalize the high degree of base selectivity exhibited by replicative DNA polymerases (pols) concur that ternary complexes formed with incorrect dNTPs are destabilized. Knowing what accounts for this destabilization is likely to be the key to understanding base discrimination. To address this issue, we have det...
Article
Full-text available
The RtcB protein has recently been identified as a 3′-phosphate RNA ligase that directly joins an RNA strand ending with a 2′,3′-cyclic phosphate to the 5′-hydroxyl group of another RNA strand in a GTP/Mn2+-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn2+ alone (RtcB/...
Article
During DNA synthesis, base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the...
Article
Residues in the nascent base pair binding pocket (NBP) of bacteriophage RB69 DNA polymerase (RB69pol) are responsible for base discrimination. Replacing Tyr567 with Ala leads to greater flexibility in the NBP, increasing the probability of misincorporation. We used the fluorescent cytosine analogue, 1,3-diaza-2-oxophenoxazine (tC(o)), to identify p...
Article
Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence...
Article
Full-text available
This article addresses the classic enigma about schizophrenia (SZ). The disease occurs with a lifetime prevalence of 1%, 80% of which is attributable to genetic factors. Females with SZ produce 50% as many children as normals, and males with SZ produce 25%. Genetic factors responsible for SZ should behave like lethal genes. Yet the prevalence of SZ...
Article
Two divalent metal ions are required for primer-extension catalyzed by DNA polymerases. One metal ion brings the 3'-hydroxyl of the primer terminus and the α-phosphorus atom of incoming dNTP together for bond formation so that the catalytically relevant conformation of the triphosphate tail of the dNTP is in an α,β,γ-tridentate coordination complex...
Article
We have recently challenged the widely held view that 2,4-difluorotoluene (dF) is a nonpolar isosteric analogue of the nucleotide dT, incapable of forming hydrogen bonds (HBs). To gain a further understanding for the kinetic preference that favors dAMP insertion opposite a templating dF, a result that mirrors the base selectivity that favors dAMP i...
Article
The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the pol...
Article
We have captured a preinsertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,β-substituted triphosphate, dUpXpp, where X is either NH or CH(2), opposite a complementary templating dA nucleotide residue. Here we report four...
Article
Results obtained using 2,4-difluorotoluene nucleobase (dF) as a nonpolar thymine isostere by Kool and colleagues challenged the Watson-Crick dogma that hydrogen bonds between complementary bases are an absolute requirement for accurate DNA replication. Here, we report crystal structure of an RB69 DNA polymerase L561A/S565G/Y567A triple mutant terna...
Article
We have previously observed that stepwise replacement of amino acid residues in the nascent base-pair binding pocket of RB69 DNA polymerase (RB69pol) with Ala or Gly expanded the space in this pocket, resulting in a progressive increase in misincorporation. However, in vivo results with similar RB69pol nascent base-pair binding pocket mutants showe...
Article
Bacteriophage RB69 DNA polymerase (RB69 pol) has served as a model for investigating how B family polymerases achieve a high level of fidelity during DNA replication. We report here the structure of an RB69 pol ternary complex at 1.8 Å resolution, extending the resolution from our previously reported structure at 2.6 Å [Franklin, M. C., et al. (200...
Article
Continuous oxidative damage inflicted on DNA produces 7,8-dihydro-8-oxoguanine (8-oxoG), a commonly occurring lesion that can potentially cause cancer by producing G → T transversions during DNA replication. Mild oxidation of 8-oxoG leads to the formation of hydantoins, specifically guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), which are...
Article
Accurate copying of the genome by DNA polymerases is challenging due in part to the continuous damage inflicted on DNA, which results from its contact with reactive oxygen species (ROS), producing lesions such as 7,8-dihydro-8-oxoguanine (8-oxoG). The deleterious effects of 8-oxoG can be attributed to its dual coding potential that leads to G --> T...
Article
Endometriosis is a major cause of chronic pain, infertility, medical and surgical interventions, and health care expenditures. Tissue factor (TF), the primary initiator of coagulation and a modulator of angiogenesis, is not normally expressed by the endothelium; however, prior studies have demonstrated that both blood vessels in solid tumors and ch...
Article
We have investigated the effect of systematically enlarging the nascent base-pair-binding pocket (NBP) of a replicative DNA polymerase from bacteriophage RB69 (RB69 pol) on the incorporation efficiency (k(pol)/K(d,app)) for both correct and incorrect dNMPs. Accordingly, we replaced residues L561, Y567, and S565 in the NBP with Ala, Ala, and Gly, re...
Article
Two divalent metal ions are required for nucleotide incorporation by DNA polymerases. Here we use the bacteriophage RB69 DNA polymerase (RB69 pol) and the metal ion exchange-inert nucleotide analogue rhodium(III) deoxythymidine triphosphate (Rh.dTTP) to investigate the requirements of metal binding to the "A" site and to the "B" site, independently...
Article
Rapid chemical quench assays, as well as equilibrium and stopped-flow fluorescence experiments, were performed with an RB69 DNA polymerase (RB69 pol)-primer-template (P/T) complex containing 2-aminopurine (dAP) and a metal exchange-inert Rh(III) derivative of a deoxynucleoside triphosphate (Rh.dTTP). The objective was to determine the effect of cat...
Article
Full-text available
Nucleic acid polymerases catalyze the formation of DNA or RNA from nucleoside-triphosphate precursors. Amino acid residues in the active site of polymerases are thought to contribute only indirectly to catalysis by serving as ligands for the two divalent cations that are required for activity or substrate binding. Two proton-transfer reactions are...
Chapter
antibiotics;acetic acid;bacitracin;molecular weight;hydrolysis
Article
Full-text available
We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol tern...
Article
Full-text available
The rate-limiting step for nucleotide incorporation in the pre-steady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D(pol)), chemistry i...
Article
Full-text available
We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/te...
Article
Full-text available
Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer stran...
Article
Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct an...
Article
To investigate the molecular basis for the selective utilization of nucleoside triphosphates complementary to templating bases, by RB69 DNA polymerase (RB69 pol), we constructed a set of mutants that we predicted would perturb the "floor" of the nascent base-pairing interface in the enzyme. We then determined the pre-steady-state kinetic parameters...
Article
Site specific mutants in the pol active center of RB69 DNA polymerase have been produced and studied using rapid chemical-quench techniques. Pre-steady-state kinetic analysis carried out with Mg(2+) and Mn(2+) has enabled us to divide the mutants into two groups. One group had greatly reduced k(pols) values in the presence of Mg(2+) but responded t...
Article
DNA polymerases from the A and B families with 3'-5' exonucleolytic activity have exonuclease domains with similar three-dimensional structures that require two divalent metal ions for catalysis. B family DNA polymerases that are part of a replicase generally have a more potent 3'-5' exonuclease (exo) activity than A family DNA polymerases that mai...
Article
Tissue factor (TF) is a transmembrane glycoprotein that initiates blood coagulation when complexed with factor (F)VIIa. Recently, TF has been shown to promote cellular signaling, tumor growth, angiogenesis, and metastasis. In the present study, we examined the pathway by which TF-FVIIa complex induces cellular signaling in human breast cancer cells...
Article
Full-text available
Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3' exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol(+) Exo(+)) enzyme, an exonuclea...
Article
Many DNA polymerases select their natural substrates, deoxy- as opposed to ribonucleoside triphosphates, with a selectivity greater than 10000-fold. The function of a highly conserved residue, Tyr416, in the palm domain of the parental enzyme, an exo(-) derivative of RB69 DNA polymerase (gp43), a member of the pol alpha DNA polymerase family, was e...
Article
Full-text available
Infection of a human melanoma cell line by a retroviral vector resulted in transmission of a mouse VL30 (mVL30-1) retroelement RNA to some of the cells infected by the retrovirus, followed by synthesis, integration, and expression of the mVL30-1 cDNA. One vector carried a tissue factor (TF) transgene that generated high TF melanoma clones, and anot...
Article
We have estimated pre-steady-state kinetic parameters for the addition of a single nucleotide residue by a set of RB69 DNA polymerase mutants in which four highly conserved residues in the fingers domain have been replaced by Ala. The relationship between the kinetic constants exhibited by the mutants and the structure of the ternary complex [Frank...
Article
Tissue factor (TF) is a transmembrane glycoprotein that complexes with factor VIIa to initiate blood coagulation. We previously reported that expression of high levels of TF in a human melanoma cell line promotes metastasis. Both the cytoplasmic domain of TF and its extracellular domain complexed with factor VIIa are required for the metastatic eff...
Article
In healthy humans, blood vessel-tissue factor is found primarily in the adventitia and thus physically separated from coagulation factors, which mainly circulate in an inactive form. Following injury, TF is exposed to blood and initiates the coagulation cascade. The resulting fibrin formation is an essential part for the initial repair of vessel da...
Article
We have determined rates for the excision of nucleotides from the 3' termini of chimeric DNA-RNA oligonucleotides using the Klenow fragment (KF) and two other DNA polymerases, from phages T4 and T7. For these studies, we synthesized DNA-RNA chimeric oligonucleotides with RNA residues in defined positions. When a ribonucleotide residue was placed at...
Article
Full-text available
The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase α class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasm...
Article
The DNA polymerase of bacteriophage T4, product of phage gene 43 (gp43), has served as a model replicative DNA polymerase in nucleic acids research for nearly 40 years. The base-selection (polymerase, or Pol) and editing (3'-exonuclease, or Exo) functions of this multifunctional protein, which have counterparts in the replicative polymerases of oth...
Article
Tissue factor (TF) is a transmembrane glycoprotein that complexes with factor VIIa to initiate blood coagulation. It was reported in an earlier study that expression of high levels of TF in a human melanoma cell line promotes metastasis, and that the cytoplasmic domain of TF is required for this metastatic effect. To analyze the functions of the cy...
Article
The function of six highly conserved residues (Arg482, Lys483, Lys486, Lys560, Asn564, and Tyr567) in the fingers domain of bacteriophage RB69 DNA polymerase (RB69 gp43) were analyzed by kinetic studies with mutants in which each of these residues was replaced with Ala. Our results suggest that Arg482, Lys486, Lys560, and Asn564 contact the incomin...
Article
We evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection...
Article
Three T4 DNA polymerase accessory proteins (44P/62P and 45P) stimulate the polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of T4 DNA polymerase (43P) on long, double-stranded DNA substrates. The 44P/62P "clamp loader" facilitates the binding of 45P, the "sliding clamp", to DNA that is primed for replication. Using a series of tru...
Article
The 2.8 A resolution crystal structure of the bacteriophage RB69 gp43, a member of the eukaryotic pol alpha family of replicative DNA polymerases, shares some similarities with other polymerases but shows many differences. Although its palm domain has the same topology as other polymerases, except rat DNA polymerase beta, one of the three carboxyla...
Article
Three groups of T4 DNA polymerase mutants were prepared and characterized. In the first group, Ala and Asn were substituted for four acidic residues in the exonuclease domain that were chosen on the basis of their sequence alignment with the Klenow fragment from Escherichia coli DNA polymerase I. Two divalent metal ions required for catalyzing the...
Article
In an attempt to define sequence elements in human and mouse tissue factor (TF) that are responsible for the species specificity observed in their interaction with human factor VIIa (HVIIa), we constructed human-mouse chimeric TF cDNAs, inserted them into plasmid vectors, and induced their expression in E. coli. Assays for procoagulant activity wer...
Article
We report the crystal structure of an NH2-terminal 388-residue fragment of T4 DNA polymerase (protein N388) refined at 2.2 A resolution. This fragment contains both the 3'-5' exonuclease active site and part of the autologous mRNA binding site (J. D. Karam, personal communication). The structure of a complex between the apoprotein N388 and a substr...
Article
Blood coagulation is initiated when tissue factor binds to coagulation factor VIIa to give an enzymatically active complex which then activates factors IX and X, leading to thrombin generation and clot formation. We have determined the crystal structure at 2.0-A degrees resolution of active-site-inhibited factor VIIa complexed with the cleaved extr...