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. 1998 Dec 1;17(23):7128–7138. doi: 10.1093/emboj/17.23.7128

Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites.

S Cal 1, K L Tan 1, A McGregor 1, B A Connolly 1
PMCID: PMC1171059  PMID: 9843516

Abstract

Bovine pancreatic deoxyribonuclease I (DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts are made between the protein and the major groove of the nucleic acid. DNase I is structurally homologous to exonuclease III, a DNA-repair enzyme with multiple activities. One of the main differences between the two enzymes is the presence of an additional alpha-helix in exonuclease III, in a position suggestive of interaction with the major groove of DNA. Recombinant DNA techniques have been used to add 14 amino acids, comprising the 10 amino acids of the exonuclease III alpha-helix flanked by a glycine rich region, to DNase I. The polypeptide has been inserted after serine 174, an amino acid on the surface of DNase I corresponding to the location of the extra alpha-helix in exonuclease III. The recombinant protein, DNase-exohelix, has been purified and its catalytic activities towards DNA investigated. The recombinant protein demonstrated a high selectivity for endonucleolytic cleavage at abasic sites in DNA, a property of exonuclease III but not native DNase I. Thus the insertion of 14 amino acids at Ser174, converts DNase I to an exonuclease III-like enzyme with DNA-repair properties.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barzilay G., Hickson I. D. Structure and function of apurinic/apyrimidinic endonucleases. Bioessays. 1995 Aug;17(8):713–719. doi: 10.1002/bies.950170808. [DOI] [PubMed] [Google Scholar]
  2. Barzilay G., Mol C. D., Robson C. N., Walker L. J., Cunningham R. P., Tainer J. A., Hickson I. D. Identification of critical active-site residues in the multifunctional human DNA repair enzyme HAP1. Nat Struct Biol. 1995 Jul;2(7):561–568. doi: 10.1038/nsb0795-561. [DOI] [PubMed] [Google Scholar]
  3. Brosius J., Holy A. Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6929–6933. doi: 10.1073/pnas.81.22.6929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brukner I., Jurukovski V., Savic A. Sequence-dependent structural variations of DNA revealed by DNase I. Nucleic Acids Res. 1990 Feb 25;18(4):891–894. doi: 10.1093/nar/18.4.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Demple B., Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem. 1994;63:915–948. doi: 10.1146/annurev.bi.63.070194.004411. [DOI] [PubMed] [Google Scholar]
  6. Doherty A. J., Connolly B. A., Worrall A. F. Overproduction of the toxic protein, bovine pancreatic DNaseI, in Escherichia coli using a tightly controlled T7-promoter-based vector. Gene. 1993 Dec 22;136(1-2):337–340. doi: 10.1016/0378-1119(93)90491-k. [DOI] [PubMed] [Google Scholar]
  7. Doherty A. J., Worrall A. F., Connolly B. A. Mutagenesis of the DNA binding residues in bovine pancreatic DNase 1: an investigation into the mechanism of sequence discrimination by a sequence selective nuclease. Nucleic Acids Res. 1991 Nov 25;19(22):6129–6132. doi: 10.1093/nar/19.22.6129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doherty A. J., Worrall A. F., Connolly B. A. The roles of arginine 41 and tyrosine 76 in the coupling of DNA recognition to phosphodiester bond cleavage by DNase I: a study using site-directed mutagenesis. J Mol Biol. 1995 Aug 18;251(3):366–377. doi: 10.1006/jmbi.1995.0440. [DOI] [PubMed] [Google Scholar]
  9. Drew H. R., Travers A. A. DNA structural variations in the E. coli tyrT promoter. Cell. 1984 Jun;37(2):491–502. doi: 10.1016/0092-8674(84)90379-9. [DOI] [PubMed] [Google Scholar]
  10. Drew H. R., Travers A. A. Structural junctions in DNA: the influence of flanking sequence on nuclease digestion specificities. Nucleic Acids Res. 1985 Jun 25;13(12):4445–4467. doi: 10.1093/nar/13.12.4445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Erzberger J. P., Barsky D., Schärer O. D., Colvin M. E., Wilson D. M., 3rd Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases. Nucleic Acids Res. 1998 Jun 1;26(11):2771–2778. doi: 10.1093/nar/26.11.2771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goljer I., Kumar S., Bolton P. H. Refined solution structure of a DNA heteroduplex containing an aldehydic abasic site. J Biol Chem. 1995 Sep 29;270(39):22980–22987. doi: 10.1074/jbc.270.39.22980. [DOI] [PubMed] [Google Scholar]
  13. Gorman M. A., Morera S., Rothwell D. G., de La Fortelle E., Mol C. D., Tainer J. A., Hickson I. D., Freemont P. S. The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites. EMBO J. 1997 Nov 3;16(21):6548–6558. doi: 10.1093/emboj/16.21.6548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gu L., Huang S. M., Sander M. Single amino acid changes alter the repair specificity of Drosophila Rrp1. Isolation of mutants deficient in repair of oxidative DNA damage. J Biol Chem. 1994 Dec 23;269(51):32685–32692. [PubMed] [Google Scholar]
  15. Harrison S. C. A structural taxonomy of DNA-binding domains. Nature. 1991 Oct 24;353(6346):715–719. doi: 10.1038/353715a0. [DOI] [PubMed] [Google Scholar]
  16. Hogan M. E., Roberson M. W., Austin R. H. DNA flexibility variation may dominate DNase I cleavage. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9273–9277. doi: 10.1073/pnas.86.23.9273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  18. Jones S. J., Worrall A. F., Connolly B. A. Site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease I. J Mol Biol. 1996 Dec 20;264(5):1154–1163. doi: 10.1006/jmbi.1996.0703. [DOI] [PubMed] [Google Scholar]
  19. KUNITZ M. Crystalline desoxyribonuclease; digestion of thymus nucleic acid; the kinetics of the reaction. J Gen Physiol. 1950 Mar;33(4):363–377. doi: 10.1085/jgp.33.4.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katayanagi K., Miyagawa M., Matsushima M., Ishikawa M., Kanaya S., Ikehara M., Matsuzaki T., Morikawa K. Three-dimensional structure of ribonuclease H from E. coli. Nature. 1990 Sep 20;347(6290):306–309. doi: 10.1038/347306a0. [DOI] [PubMed] [Google Scholar]
  21. Lahm A., Suck D. DNase I-induced DNA conformation. 2 A structure of a DNase I-octamer complex. J Mol Biol. 1991 Dec 5;222(3):645–667. doi: 10.1016/0022-2836(91)90502-w. [DOI] [PubMed] [Google Scholar]
  22. Liao T. Deoxythymidine 3', 5'-di-p-nitrophenyl phosphate as a synthetic substrate for bovine pancreatic deoxyribonuclease. J Biol Chem. 1975 May 25;250(10):3721–3724. [PubMed] [Google Scholar]
  23. Lindahl T., Ljungquist S., Siegert W., Nyberg B., Sperens B. DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli. J Biol Chem. 1977 May 25;252(10):3286–3294. [PubMed] [Google Scholar]
  24. Lomonossoff G. P., Butler P. J., Klug A. Sequence-dependent variation in the conformation of DNA. J Mol Biol. 1981 Jul 15;149(4):745–760. doi: 10.1016/0022-2836(81)90356-9. [DOI] [PubMed] [Google Scholar]
  25. McGregor A., Rao M. V., Duckworth G., Stockley P. G., Connolly B. A. Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation. Nucleic Acids Res. 1996 Aug 15;24(16):3173–3180. doi: 10.1093/nar/24.16.3173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mendoza R., Markovits J., Jaffrezou J. P., Muzard G., Le Pecq J. B. DNase I susceptibility of bent DNA and its alteration by ditercalinium and distamycin. Biochemistry. 1990 May 29;29(21):5035–5043. doi: 10.1021/bi00473a006. [DOI] [PubMed] [Google Scholar]
  27. Mol C. D., Kuo C. F., Thayer M. M., Cunningham R. P., Tainer J. A. Structure and function of the multifunctional DNA-repair enzyme exonuclease III. Nature. 1995 Mar 23;374(6520):381–386. doi: 10.1038/374381a0. [DOI] [PubMed] [Google Scholar]
  28. Oefner C., Suck D. Crystallographic refinement and structure of DNase I at 2 A resolution. J Mol Biol. 1986 Dec 5;192(3):605–632. doi: 10.1016/0022-2836(86)90280-9. [DOI] [PubMed] [Google Scholar]
  29. Pabo C. O., Sauer R. T. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095. doi: 10.1146/annurev.bi.61.070192.005201. [DOI] [PubMed] [Google Scholar]
  30. Price P. A. Characterization of Ca ++ and Mg ++ binding to bovine pancreatic deoxyribonuclease A. J Biol Chem. 1972 May 10;247(9):2895–2899. [PubMed] [Google Scholar]
  31. Price P. A., Liu T. Y., Stein W. H., Moore S. Properties of chromatographically purified bovine pancreatic deoxyribonuclease. J Biol Chem. 1969 Feb 10;244(3):917–923. [PubMed] [Google Scholar]
  32. Price P. A., Stein W. H., Moore S. Effect of divalent cations on the reduction and re-formation of the disulfide bonds of deoxyribonuclease. J Biol Chem. 1969 Feb 10;244(3):929–932. [PubMed] [Google Scholar]
  33. Price P. A. The essential role of Ca2+ in the activity of bovine pancreatic deoxyribonuclease. J Biol Chem. 1975 Mar 25;250(6):1981–1986. [PubMed] [Google Scholar]
  34. Rogers S. G., Weiss B. Exonuclease III of Escherichia coli K-12, an AP endonuclease. Methods Enzymol. 1980;65(1):201–211. doi: 10.1016/s0076-6879(80)65028-9. [DOI] [PubMed] [Google Scholar]
  35. Sadowski P. D., Hurwitz J. Enzymatic breakage of deoxyribonucleic acid. II. Purification and properties of endonuclease IV from T4 phage-infected Escherichia coli. J Biol Chem. 1969 Nov 25;244(22):6192–6198. [PubMed] [Google Scholar]
  36. Seeman N. C., Rosenberg J. M., Rich A. Sequence-specific recognition of double helical nucleic acids by proteins. Proc Natl Acad Sci U S A. 1976 Mar;73(3):804–808. doi: 10.1073/pnas.73.3.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shida T., Noda M., Sekiguchi J. Cleavage of single- and double-stranded DNAs containing an abasic residue by Escherichia coli exonuclease III (AP endonuclease VI). Nucleic Acids Res. 1996 Nov 15;24(22):4572–4576. doi: 10.1093/nar/24.22.4572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Steitz T. A. Structural studies of protein-nucleic acid interaction: the sources of sequence-specific binding. Q Rev Biophys. 1990 Aug;23(3):205–280. doi: 10.1017/s0033583500005552. [DOI] [PubMed] [Google Scholar]
  39. Suck D., Lahm A., Oefner C. Structure refined to 2A of a nicked DNA octanucleotide complex with DNase I. Nature. 1988 Mar 31;332(6163):464–468. doi: 10.1038/332464a0. [DOI] [PubMed] [Google Scholar]
  40. Suck D., Oefner C., Kabsch W. Three-dimensional structure of bovine pancreatic DNase I at 2.5 A resolution. EMBO J. 1984 Oct;3(10):2423–2430. doi: 10.1002/j.1460-2075.1984.tb02149.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Suck D., Oefner C. Structure of DNase I at 2.0 A resolution suggests a mechanism for binding to and cutting DNA. Nature. 1986 Jun 5;321(6070):620–625. doi: 10.1038/321620a0. [DOI] [PubMed] [Google Scholar]
  42. Warren M. A., Evans S. J., Connolly B. A. Effects of non-conservative changes to tyrosine 76, a key DNA binding residue of DNase I, on phosphodiester bond cleavage and DNA hydrolysis selectivity. Protein Eng. 1997 Mar;10(3):279–283. doi: 10.1093/protein/10.3.279. [DOI] [PubMed] [Google Scholar]
  43. Weston S. A., Lahm A., Suck D. X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution. J Mol Biol. 1992 Aug 20;226(4):1237–1256. doi: 10.1016/0022-2836(92)91064-v. [DOI] [PubMed] [Google Scholar]
  44. Wolf E., Brukner I., Suck D. Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity. Protein Eng. 1995 Mar;8(3):283–291. doi: 10.1093/protein/8.3.283. [DOI] [PubMed] [Google Scholar]
  45. Worrall A. F., Connolly B. A. The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli. J Biol Chem. 1990 Dec 15;265(35):21889–21895. [PubMed] [Google Scholar]
  46. Yang W., Steitz T. A. Recombining the structures of HIV integrase, RuvC and RNase H. Structure. 1995 Feb 15;3(2):131–134. doi: 10.1016/s0969-2126(01)00142-3. [DOI] [PubMed] [Google Scholar]

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