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. 2001 Dec;81(6):3545–3559. doi: 10.1016/S0006-3495(01)75985-1

Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: optical absorbance and fluorescence studies.

A V Cherepanov 1, S de Vries 1
PMCID: PMC1301809  PMID: 11721015

Abstract

The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the K(d) between 0.1 and 20 microM. Nucleotide binding results in a decrease of absorbance at 260 nm due to pi-stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the pi-stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K(d) for the noncovalent site is approximately 1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity.

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Selected References

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  1. Arabshahi A., Frey P. A. Standard free energy for the hydrolysis of adenylylated T4 DNA ligase and the apparent pKa of lysine 159. J Biol Chem. 1999 Mar 26;274(13):8586–8588. doi: 10.1074/jbc.274.13.8586. [DOI] [PubMed] [Google Scholar]
  2. Aravind L., Koonin E. V. DNA polymerase beta-like nucleotidyltransferase superfamily: identification of three new families, classification and evolutionary history. Nucleic Acids Res. 1999 Apr 1;27(7):1609–1618. doi: 10.1093/nar/27.7.1609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Atencia E. A., Madrid O., Günther Sillero M. A., Sillero A. T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates. Eur J Biochem. 1999 May;261(3):802–811. doi: 10.1046/j.1432-1327.1999.00338.x. [DOI] [PubMed] [Google Scholar]
  4. Burstein E. A., Vedenkina N. S., Ivkova M. N. Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol. 1973 Oct;18(4):263–279. doi: 10.1111/j.1751-1097.1973.tb06422.x. [DOI] [PubMed] [Google Scholar]
  5. CHARNEY E., GELLERT M. SOLVENT-INDUCED ULTRAVIOLET HYPOCHROMISM IN NUCLEOSIDES. Biopolym Symp. 1964;13:469–477. [PubMed] [Google Scholar]
  6. Callis P. R. 1La and 1Lb transitions of tryptophan: applications of theory and experimental observations to fluorescence of proteins. Methods Enzymol. 1997;278:113–150. doi: 10.1016/s0076-6879(97)78009-1. [DOI] [PubMed] [Google Scholar]
  7. DEVOE H., TINOCO I., Jr The hypochromism of helical polynucleotides. J Mol Biol. 1962 Jun;4:518–527. doi: 10.1016/s0022-2836(62)80106-5. [DOI] [PubMed] [Google Scholar]
  8. Delabar J. M., Guschlbauer W., Schneider C., Thiéry J. Nucleoside conformations. VII. Solvent effects on optical properties of adenosine and its derivatives in dilute solutions. Biochimie. 1972;54(8):1041–1048. doi: 10.1016/s0300-9084(72)80055-5. [DOI] [PubMed] [Google Scholar]
  9. Doherty A. J., Ashford S. R., Subramanya H. S., Wigley D. B. Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization. J Biol Chem. 1996 May 10;271(19):11083–11089. doi: 10.1074/jbc.271.19.11083. [DOI] [PubMed] [Google Scholar]
  10. Herskovits T. T., Sorensen M. Studies of the location of tyrosyl and tryptophyl residues in protein. II. Applications of model data to solvent perturbation studies of proteins rich in both tyrosine and tryptophan. Biochemistry. 1968 Jul;7(7):2533–2542. doi: 10.1021/bi00847a013. [DOI] [PubMed] [Google Scholar]
  11. Håkansson K., Doherty A. J., Shuman S., Wigley D. B. X-ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzymes. Cell. 1997 May 16;89(4):545–553. doi: 10.1016/s0092-8674(00)80236-6. [DOI] [PubMed] [Google Scholar]
  12. Ishida T., Usami H., Inoue M., Yamagata Y., Tomita K. U. The stacking interaction in 9-(indole-3-propyl)-1-methyl adeninium iodide crystal, a model study on the interaction between tryptophan residue and adenine base in protein-nucleic acid interactions. Biochem Biophys Res Commun. 1982 Jul 30;107(2):746–751. doi: 10.1016/0006-291x(82)91554-6. [DOI] [PubMed] [Google Scholar]
  13. Kachurin A. M., Smelianskii A. Ia, Chernaenko V. M., Akhmedov A. T. Vzaimodeistvie belka RecA s ADP (ATP): k voprosu o mekhanizme ATP-aznoi reaktsii. Mol Biol (Mosk) 1990 May-Jun;24(3):621–628. [PubMed] [Google Scholar]
  14. Kondo N. S., Holmes H. M., Stempel L. M., Ts'o O. P. Influence of the phosphodiester linkage (3'-5', 2'-5', and 5'-5') on the conformation of dinucleoside monophosphate. Biochemistry. 1970 Sep 1;9(18):3479–3498. doi: 10.1021/bi00820a002. [DOI] [PubMed] [Google Scholar]
  15. Koonin E. V., Gorbalenya A. E. Related domains in yeast tRNA ligase, bacteriophage T4 polynucleotide kinase and RNA ligase, and mammalian myelin 2',3'-cyclic nucleotide phosphohydrolase revealed by amino acid sequence comparison. FEBS Lett. 1990 Jul 30;268(1):231–234. doi: 10.1016/0014-5793(90)81015-g. [DOI] [PubMed] [Google Scholar]
  16. Lee J. Y., Chang C., Song H. K., Moon J., Yang J. K., Kim H. K., Kwon S. T., Suh S. W. Crystal structure of NAD(+)-dependent DNA ligase: modular architecture and functional implications. EMBO J. 2000 Mar 1;19(5):1119–1129. doi: 10.1093/emboj/19.5.1119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lin S. W., Sakmar T. P. Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state. Biochemistry. 1996 Aug 27;35(34):11149–11159. doi: 10.1021/bi960858u. [DOI] [PubMed] [Google Scholar]
  18. Madrid O., Martín D., Atencia E. A., Sillero A., Günther Sillero M. A. T4 DNA ligase synthesizes dinucleoside polyphosphates. FEBS Lett. 1998 Aug 21;433(3):283–286. doi: 10.1016/s0014-5793(98)00932-6. [DOI] [PubMed] [Google Scholar]
  19. Modorich P., Lehman I. R. Deoxyribonucleic acid ligase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from Escherichia coli. J Biol Chem. 1973 Nov 10;248(21):7502–7511. [PubMed] [Google Scholar]
  20. Montecucco A., Lestingi M., Pedrali-Noy G., Spadari S., Ciarrocchi G. Use of ATP, dATP and their alpha-thio derivatives to study DNA ligase adenylation. Biochem J. 1990 Oct 1;271(1):265–268. doi: 10.1042/bj2710265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Morita F. Molecular complex of tryptophan with ATP or its analogs. Biochim Biophys Acta. 1974 May 24;343(3):674–681. doi: 10.1016/0304-4165(74)90288-8. [DOI] [PubMed] [Google Scholar]
  22. Odell M., Sriskanda V., Shuman S., Nikolov D. B. Crystal structure of eukaryotic DNA ligase-adenylate illuminates the mechanism of nick sensing and strand joining. Mol Cell. 2000 Nov;6(5):1183–1193. doi: 10.1016/s1097-2765(00)00115-5. [DOI] [PubMed] [Google Scholar]
  23. Pörschke D. Structure and dynamics of a tryptophanepeptide-polynucleotide complex. Nucleic Acids Res. 1980 Apr 11;8(7):1591–1612. doi: 10.1093/nar/8.7.1591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Raae A. J., Kleppe R. K., Kleppe K. Kinetics and effect of salts and polyamines on T4 polynucleotide ligase. Eur J Biochem. 1975 Dec 15;60(2):437–443. doi: 10.1111/j.1432-1033.1975.tb21021.x. [DOI] [PubMed] [Google Scholar]
  25. Shatkin A. J. Capping of eucaryotic mRNAs. Cell. 1976 Dec;9(4 Pt 2):645–653. doi: 10.1016/0092-8674(76)90128-8. [DOI] [PubMed] [Google Scholar]
  26. Shimizu T., Morii H. Spectroscopic studies of the ncd motor domain.ADP complex: CD spectrum of ADP induced by binding to the motor domain of ncd. Biochemistry. 1998 Nov 24;37(47):16680–16685. doi: 10.1021/bi981338x. [DOI] [PubMed] [Google Scholar]
  27. Shuman S., Schwer B. RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases. Mol Microbiol. 1995 Aug;17(3):405–410. doi: 10.1111/j.1365-2958.1995.mmi_17030405.x. [DOI] [PubMed] [Google Scholar]
  28. Silber R., Malathi V. G., Hurwitz J. Purification and properties of bacteriophage T4-induced RNA ligase. Proc Natl Acad Sci U S A. 1972 Oct;69(10):3009–3013. doi: 10.1073/pnas.69.10.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sillero A., Sillero M. A. Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases. Pharmacol Ther. 2000 Aug-Sep;87(2-3):91–102. doi: 10.1016/s0163-7258(00)00047-4. [DOI] [PubMed] [Google Scholar]
  30. Sokolov N. N. DNK- i RNK-ligazy. Biokhimiia. 1976 Sep;41(9):1722–1725. [PubMed] [Google Scholar]
  31. Strickland E. H., Billups C., Kay E. Effects of hydrogen bonding and solvents upon the tryptophanyl 1 L a absorption band. Studies using 2,3-dimethylindole. Biochemistry. 1972 Sep 12;11(19):3657–3662. doi: 10.1021/bi00769a025. [DOI] [PubMed] [Google Scholar]
  32. Strickland E. H., Horwitz J., Kay E., Shannon L. M., Wilchek M., Billups C. Near-ultraviolet absorption bands of tryptophan. Studies using horseradish peroxidase isoenzymes, bovine and horse heart cytochrome c, and N-stearyl-L-tryptophan n-hexyl ester. Biochemistry. 1971 Jun 22;10(13):2631–2638. doi: 10.1021/bi00789a033. [DOI] [PubMed] [Google Scholar]
  33. Subramanya H. S., Doherty A. J., Ashford S. R., Wigley D. B. Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7. Cell. 1996 May 17;85(4):607–615. doi: 10.1016/s0092-8674(00)81260-x. [DOI] [PubMed] [Google Scholar]
  34. Timson D. J., Singleton M. R., Wigley D. B. DNA ligases in the repair and replication of DNA. Mutat Res. 2000 Aug 30;460(3-4):301–318. doi: 10.1016/s0921-8777(00)00033-1. [DOI] [PubMed] [Google Scholar]
  35. Toulme J. J. Stacking interactions between aromatic amino acids and adenine ring of ATP in zinc mediated ternary complexes. Bioinorg Chem. 1978 Apr;8(4):319–329. doi: 10.1016/s0006-3061(00)80165-9. [DOI] [PubMed] [Google Scholar]
  36. Vivian J. T., Callis P. R. Mechanisms of tryptophan fluorescence shifts in proteins. Biophys J. 2001 May;80(5):2093–2109. doi: 10.1016/S0006-3495(01)76183-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Volkov S. N., Pechenaia V. I. O vliianii konformatsionnykh izmenenii dvoinoi spiralia na gipokhromism DNK. Mol Biol (Mosk) 1982 Mar-Apr;16(2):322–329. [PubMed] [Google Scholar]
  38. Weiss B., Jacquemin-Sablon A., Live T. R., Fareed G. C., Richardson C. C. Enzymatic breakage and joining of deoxyribonucleic acid. VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4. J Biol Chem. 1968 Sep 10;243(17):4543–4555. [PubMed] [Google Scholar]
  39. Weiss B., Thompson A., Richardson C. C. Ezymatic breakage and joining of deoxyribonucleic acid. VII. Properties of the enzyme-adenylate intermediate in the polynucleotide ligase reaction. J Biol Chem. 1968 Sep 10;243(17):4556–4563. [PubMed] [Google Scholar]
  40. West J. J. Adenosine triphosphate and inosine triphosphate dependent conformational changes of adenosine diphosphate-G-actin. Biochemistry. 1970 Sep 29;9(20):3847–3853. doi: 10.1021/bi00822a001. [DOI] [PubMed] [Google Scholar]
  41. YANARI S., BOVEY F. A. Interpretation of the ultraviolet spectral changes of proteins. J Biol Chem. 1960 Oct;235:2818–2826. [PubMed] [Google Scholar]
  42. Zagrebel'nyi S. N., Zernov Iu P., Knorre D. G. Kineticheskie kharakteristiki izotopnogo obmena ATP-pirofosfat, kataliziruemogo RNK-ligazoi bakteriofaga T4. Mol Biol (Mosk) 1984 Jan-Feb;18(1):227–233. [PubMed] [Google Scholar]

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