Bill Konigsberg, PhD
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Research Summary
The current objective of our laboratory is to determine the mechanisms used by B family DNA polymerases for base discrimination. We have selected a T even phage DNA polymerase, RB69 pol, as a prototype for several reasons: (i) it has considerable sequence similarities to human DNA pol a and d; (ii) it is assembled into a DNA replicase with accessory proteins in an analogous fashion to human DNA replicases; (iii) there is a wealth of structural and kinetic information about this enzyme that enables new findings about the mechanism of action of RB69 pol to be interpreted within a well established framework; (iv) despite extensive studies it is still unclear how RB69 as well as other DNA pols are able to replicate DNA with a minimum of errors (2x10-8 per base per genome replication).
Specialized Terms: Blood Coagulation; DNA Replication; Genetics; Proteins and Macromolecules
Extensive Research Description
Molecular Biology and Structure of a DNA Replicase
The current objective of our laboratory is to determine the mechanisms used by B family DNA polymerases for base discrimination. We have selected a T even phage DNA polymerase, RB69 pol, as a prototype for several reasons: (i) it has considerable sequence similarities to human DNA pol a and d; (ii) it is assembled into a DNA replicase with accessory proteins in an analogous fashion to human DNA replicases; (iii) there is a wealth of structural and kinetic information about this enzyme that enables new findings about the mechanism of action of RB69 pol to be interpreted within a well established framework; (iv) despite extensive studies it is still unclear how RB69 as well as other DNA pols are able to replicate DNA with a minimum of errors (2x10-8 per base per genome replication). To understand how this level of fidelity is achieved we are investigating the kinetic behavior of the wild type and selected RB69 polymerase mutants complexed with short dsDNA fragments (P/T) when challenged with correct or with non-complementary dNTPs. Among the wide variety of experimental approaches that we are using to investigate the mechanisms employed by RB69 pol for faithful DNA replication are: (i) rapid chemical quench to estimate pre-steady-state kinetic parameters for wild type and RB69 pol mutants with amino acid substitutions that affect fidelity; (ii) stopped-flow fluorescence to determine rates of conformational changes during the polymerization and editing cycles; (iii) single molecule fluorescence and single molecule FRET to monitor the dynamics of the nucleotidyl transfer reaction and to detect conformationally distinct transient intermediates that may have a profound influence on fidelity. (iv) x-ray crystallography to determine the structure of RB69 pol mutants that display dramatically reduced base discrimination while maintaining pre-steady-state kinetic parameters for incorporation of correct dNMPs that match those of the wild type enzyme. From the results of these experiments we hope to be able to identify the rate limiting step for the incorporation of correct and incorrect bases.
Ultimately we hope to assemble the complete DNA replication complex, and elucidate the mechanisms that are responsible for the faithful replication of the T even phage genome. We believe that detailed information about this system will be valuable in deciphering the enzymology of viral and mammalian replication systems.Research Interests
Biochemistry; Biophysics; Blood Coagulation; DNA Replication; Genetics; Molecular Biology
Selected Publications
- The covalent structure of a human gamma G-immunoglobulin. 8. Amino acid sequence of heavy-chain cyanogen bromide fragments H5-H7.Rutishauser U, Cunningham B, Bennett C, Konigsberg W, Edelman G. The covalent structure of a human gamma G-immunoglobulin. 8. Amino acid sequence of heavy-chain cyanogen bromide fragments H5-H7. Biochemistry 1970, 9: 3171-81. PMID: 5530842, DOI: 10.1021/bi00818a009.
- The covalent structure of a human gamma G-immunoglobulin. IX. Assignment of asparaginyl and glutaminyl residues.Bennett C, Konigsberg W, Edelman G. The covalent structure of a human gamma G-immunoglobulin. IX. Assignment of asparaginyl and glutaminyl residues. Biochemistry 1970, 9: 3181-8. PMID: 5489772, DOI: 10.1021/bi00818a010.
- Amino acid sequence of the Fc region of a human gamma G-immunoglobulin.Rutishauser U, Cunningham B, Bennett C, Konigsberg W, Edelman G. Amino acid sequence of the Fc region of a human gamma G-immunoglobulin. Proceedings Of The National Academy Of Sciences Of The United States Of America 1968, 61: 1414-1421. PMID: 5249817, PMCID: PMC225271, DOI: 10.1073/pnas.61.4.1414.
- Variable regions of heavy and light polypeptide chains of the same gammaG-immunoglobulin molecule.Gottlieb P, Cunningham B, Waxdal M, Konigsberg W, Edelman G. Variable regions of heavy and light polypeptide chains of the same gammaG-immunoglobulin molecule. Proceedings Of The National Academy Of Sciences Of The United States Of America 1968, 61: 168-175. PMID: 4177258, PMCID: PMC285919, DOI: 10.1073/pnas.61.1.168.
- The covalent structure of a human gamma G-immunoglobulin. I. Isolation and characterization of the whole molecule, the polypeptide chains, and the tryptic fragments.Edelman G, Gall W, Waxdal M, Konigsberg W. The covalent structure of a human gamma G-immunoglobulin. I. Isolation and characterization of the whole molecule, the polypeptide chains, and the tryptic fragments. Biochemistry 1968, 7: 1950-8. PMID: 4967805, DOI: 10.1021/bi00845a045.
- The covalent structure of a human gamma G-immunoglobulin. IV. The interchain disulfide bonds.Gall W, Cunningham B, Waxdal M, Konigsberg W, Edelman G. The covalent structure of a human gamma G-immunoglobulin. IV. The interchain disulfide bonds. Biochemistry 1968, 7: 1973-82. PMID: 5650388, DOI: 10.1021/bi00845a048.
- The covalent structure of a human gamma G-immunoglobulin. V. Partial amino acid sequence of the light chain.Cunningham B, Gottlieb P, Konigsberg W, Edelman G. The covalent structure of a human gamma G-immunoglobulin. V. Partial amino acid sequence of the light chain. Biochemistry 1968, 7: 1983-94. PMID: 5650389, DOI: 10.1021/bi00845a049.
- The covalent structure of a human gamma G-immunoglobulin. II. Isolation and characterization of the cyanogen bromide fragments.Waxdal M, Konigsberg W, Henley W, Edelman G. The covalent structure of a human gamma G-immunoglobulin. II. Isolation and characterization of the cyanogen bromide fragments. Biochemistry 1968, 7: 1959-66. PMID: 5650387, DOI: 10.1021/bi00845a046.
- The covalent structure of a human gamma G-immunoglobulin. 3. Arrangement of the cyanogen bromide fragments.Waxdal M, Konigsberg W, Edelman G. The covalent structure of a human gamma G-immunoglobulin. 3. Arrangement of the cyanogen bromide fragments. Biochemistry 1968, 7: 1967-72. PMID: 5655436, DOI: 10.1021/bi00845a047.
- The Structure of a Human Gamma G ImmunoglobulinWaxdal M, Konigsberg W, Edelman G. The Structure of a Human Gamma G Immunoglobulin. Cold Spring Harbor Symposia On Quantitative Biology 1967, 32: 53-63. DOI: 10.1101/sqb.1967.032.01.010.