Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1995 Nov 15;14(22):5724–5735. doi: 10.1002/j.1460-2075.1995.tb00259.x

Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site.

J E Mueller 1, D Smith 1, M Bryk 1, M Belfort 1
PMCID: PMC394687  PMID: 8521829

Abstract

I-TevI, the intron-encoded endonuclease from the thymidylate synthase (td) gene of bacteriophage T4, binds its DNA substrate across the minor groove in a sequence-tolerant fashion. We demonstrate here that the 28 kDa I-TevI binds the extensive 37 bp td homing site as a monomer and significantly distorts its substrate. In situ cleavage assays and phasing analyses indicate that upon nicking the bottom strand of the td homing site, I-TevI induces a directed bend of 38 degrees towards the major groove near the cleavage site. Formation of the bent I-TevI-DNA complex is proposed to promote top-strand cleavage of the homing site. Furthermore, reductions in the degree of distortion and in the efficiency of binding base-substitution variants of the td homing site indicate that sequences flanking the cleavage site contribute to the I-TevI-induced conformational change. These results, combined with genetic, physical and computer-modeling studies, form the basis of a model, wherein I-TevI acts as a hinged monomer to induce a distortion that widens the minor groove, facilitating access to the top-strand cleavage site. The model is compatible with both unmodified DNA and glucosylated hydroxymethylcytosine-containing DNA, as exists in the T-even phages.

Full text

PDF
5728

Images in this article

5725Image
on p.5725
5726Image
on p.5726
5728Image
on p.5728
5729Image
on p.5729
5730Image
on p.5730
5731Image
on p.5731

Selected References

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

  1. Avitahl N., Calame K. The C/EBP family of proteins distorts DNA upon binding but does not introduce a large directed bend. J Biol Chem. 1994 Sep 23;269(38):23553–23562. [PubMed] [Google Scholar]
  2. Aymami J., Coll M., van der Marel G. A., van Boom J. H., Wang A. H., Rich A. Molecular structure of nicked DNA: a substrate for DNA repair enzymes. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2526–2530. doi: 10.1073/pnas.87.7.2526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Belfort M. Phage T4 introns: self-splicing and mobility. Annu Rev Genet. 1990;24:363–385. doi: 10.1146/annurev.ge.24.120190.002051. [DOI] [PubMed] [Google Scholar]
  4. Bell-Pedersen D., Quirk S. M., Aubrey M., Belfort M. A site-specific endonuclease and co-conversion of flanking exons associated with the mobile td intron of phage T4. Gene. 1989 Oct 15;82(1):119–126. doi: 10.1016/0378-1119(89)90036-x. [DOI] [PubMed] [Google Scholar]
  5. Bell-Pedersen D., Quirk S. M., Bryk M., Belfort M. I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7719–7723. doi: 10.1073/pnas.88.17.7719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bell-Pedersen D., Quirk S., Clyman J., Belfort M. Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications. Nucleic Acids Res. 1990 Jul 11;18(13):3763–3770. doi: 10.1093/nar/18.13.3763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bryk M., Belisle M., Mueller J. E., Belfort M. Selection of a remote cleavage site by I-tevI, the td intron-encoded endonuclease. J Mol Biol. 1995 Mar 24;247(2):197–210. doi: 10.1006/jmbi.1994.0133. [DOI] [PubMed] [Google Scholar]
  8. Bryk M., Quirk S. M., Mueller J. E., Loizos N., Lawrence C., Belfort M. The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target. EMBO J. 1993 May;12(5):2141–2149. doi: 10.1002/j.1460-2075.1993.tb05862.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chu F. K., Maley G., Pedersen-Lane J., Wang A. M., Maley F. Characterization of the restriction site of a prokaryotic intron-encoded endonuclease. Proc Natl Acad Sci U S A. 1990 May;87(9):3574–3578. doi: 10.1073/pnas.87.9.3574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clyman J., Belfort M. Trans and cis requirements for intron mobility in a prokaryotic system. Genes Dev. 1992 Jul;6(7):1269–1279. doi: 10.1101/gad.6.7.1269. [DOI] [PubMed] [Google Scholar]
  11. FERGUSON K. A. STARCH-GEL ELECTROPHORESIS--APPLICATION TO THE CLASSIFICATION OF PITUITARY PROTEINS AND POLYPEPTIDES. Metabolism. 1964 Oct;13:SUPPL–SUPPL1002. doi: 10.1016/s0026-0495(64)80018-4. [DOI] [PubMed] [Google Scholar]
  12. Feng J. A., Johnson R. C., Dickerson R. E. Hin recombinase bound to DNA: the origin of specificity in major and minor groove interactions. Science. 1994 Jan 21;263(5145):348–355. doi: 10.1126/science.8278807. [DOI] [PubMed] [Google Scholar]
  13. Flick K. E., Gonzalez L., Jr, Harrison C. J., Nelson H. C. Yeast heat shock transcription factor contains a flexible linker between the DNA-binding and trimerization domains. Implications for DNA binding by trimeric proteins. J Biol Chem. 1994 Apr 29;269(17):12475–12481. [PubMed] [Google Scholar]
  14. Gartenberg M. R., Crothers D. M. DNA sequence determinants of CAP-induced bending and protein binding affinity. Nature. 1988 Jun 30;333(6176):824–829. doi: 10.1038/333824a0. [DOI] [PubMed] [Google Scholar]
  15. Giese K., Cox J., Grosschedl R. The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell. 1992 Apr 3;69(1):185–195. doi: 10.1016/0092-8674(92)90129-z. [DOI] [PubMed] [Google Scholar]
  16. Halford S. E., Goodall A. J. Modes of DNA cleavage by the EcoRV restriction endonuclease. Biochemistry. 1988 Mar 8;27(5):1771–1777. doi: 10.1021/bi00405a058. [DOI] [PubMed] [Google Scholar]
  17. Hope I. A., Struhl K. GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J. 1987 Sep;6(9):2781–2784. doi: 10.1002/j.1460-2075.1987.tb02573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kahn J. D., Yun E., Crothers D. M. Detection of localized DNA flexibility. Nature. 1994 Mar 10;368(6467):163–166. doi: 10.1038/368163a0. [DOI] [PubMed] [Google Scholar]
  19. Kerppola T. K., Curran T. DNA bending by Fos and Jun: the flexible hinge model. Science. 1991 Nov 22;254(5035):1210–1214. doi: 10.1126/science.1957173. [DOI] [PubMed] [Google Scholar]
  20. Kerppola T. K., Curran T. Fos-Jun heterodimers and Jun homodimers bend DNA in opposite orientations: implications for transcription factor cooperativity. Cell. 1991 Jul 26;66(2):317–326. doi: 10.1016/0092-8674(91)90621-5. [DOI] [PubMed] [Google Scholar]
  21. Kim J. L., Nikolov D. B., Burley S. K. Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature. 1993 Oct 7;365(6446):520–527. doi: 10.1038/365520a0. [DOI] [PubMed] [Google Scholar]
  22. Kim J., Zwieb C., Wu C., Adhya S. Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector. Gene. 1989 Dec 21;85(1):15–23. doi: 10.1016/0378-1119(89)90459-9. [DOI] [PubMed] [Google Scholar]
  23. Kim Y., Geiger J. H., Hahn S., Sigler P. B. Crystal structure of a yeast TBP/TATA-box complex. Nature. 1993 Oct 7;365(6446):512–520. doi: 10.1038/365512a0. [DOI] [PubMed] [Google Scholar]
  24. King C. Y., Weiss M. A. The SRY high-mobility-group box recognizes DNA by partial intercalation in the minor groove: a topological mechanism of sequence specificity. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11990–11994. doi: 10.1073/pnas.90.24.11990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Klemm J. D., Rould M. A., Aurora R., Herr W., Pabo C. O. Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell. 1994 Apr 8;77(1):21–32. doi: 10.1016/0092-8674(94)90231-3. [DOI] [PubMed] [Google Scholar]
  26. Koo H. S., Drak J., Rice J. A., Crothers D. M. Determination of the extent of DNA bending by an adenine-thymine tract. Biochemistry. 1990 May 1;29(17):4227–4234. doi: 10.1021/bi00469a027. [DOI] [PubMed] [Google Scholar]
  27. Le Cam E., Fack F., Ménissier-de Murcia J., Cognet J. A., Barbin A., Sarantoglou V., Révet B., Delain E., de Murcia G. Conformational analysis of a 139 base-pair DNA fragment containing a single-stranded break and its interaction with human poly(ADP-ribose) polymerase. J Mol Biol. 1994 Jan 21;235(3):1062–1071. doi: 10.1006/jmbi.1994.1057. [DOI] [PubMed] [Google Scholar]
  28. Lerman L. S., Frisch H. L. Why does the electrophoretic mobility of DNA in gels vary with the length of the molecule? Biopolymers. 1982 May;21(5):995–997. doi: 10.1002/bip.360210511. [DOI] [PubMed] [Google Scholar]
  29. Li L., Chandrasegaran S. Alteration of the cleavage distance of Fok I restriction endonuclease by insertion mutagenesis. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2764–2768. doi: 10.1073/pnas.90.7.2764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li L., Wu L. P., Chandrasegaran S. Functional domains in Fok I restriction endonuclease. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4275–4279. doi: 10.1073/pnas.89.10.4275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu-Johnson H. N., Gartenberg M. R., Crothers D. M. The DNA binding domain and bending angle of E. coli CAP protein. Cell. 1986 Dec 26;47(6):995–1005. doi: 10.1016/0092-8674(86)90814-7. [DOI] [PubMed] [Google Scholar]
  32. Lumpkin O. J. Mobility of DNA in gel electrophoresis. Biopolymers. 1982 Nov;21(11):2315–2316. doi: 10.1002/bip.360211116. [DOI] [PubMed] [Google Scholar]
  33. Mills J. B., Cooper J. P., Hagerman P. J. Electrophoretic evidence that single-stranded regions of one or more nucleotides dramatically increase the flexibility of DNA. Biochemistry. 1994 Feb 22;33(7):1797–1803. doi: 10.1021/bi00173a024. [DOI] [PubMed] [Google Scholar]
  34. Orchard K., May G. E. An EMSA-based method for determining the molecular weight of a protein--DNA complex. Nucleic Acids Res. 1993 Jul 11;21(14):3335–3336. doi: 10.1093/nar/21.14.3335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pieters J. M., Mans R. M., van den Elst H., van der Marel G. A., van Boom J. H., Altona C. Conformational and thermodynamic consequences of the introduction of a nick in duplexed DNA fragments: an NMR study augmented by biochemical experiments. Nucleic Acids Res. 1989 Jun 26;17(12):4551–4565. doi: 10.1093/nar/17.12.4551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Robertson C. A., Nash H. A. Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor. J Biol Chem. 1988 Mar 15;263(8):3554–3557. [PubMed] [Google Scholar]
  37. Salvo J. J., Grindley N. D. The gamma delta resolvase bends the res site into a recombinogenic complex. EMBO J. 1988 Nov;7(11):3609–3616. doi: 10.1002/j.1460-2075.1988.tb03239.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sayre M. H., Geiduschek E. P. Effects of mutations at amino acid 61 in the arm of TF1 on its DNA-binding properties. J Mol Biol. 1990 Dec 20;216(4):819–833. doi: 10.1016/S0022-2836(99)80004-7. [DOI] [PubMed] [Google Scholar]
  39. Schneider G. J., Sayre M. H., Geiduschek E. P. DNA-bending properties of TF1. J Mol Biol. 1991 Oct 5;221(3):777–794. doi: 10.1016/0022-2836(91)80175-t. [DOI] [PubMed] [Google Scholar]
  40. Strzelecka T. E., Hayes J. J., Clore G. M., Gronenborn A. M. DNA binding specificity of the Mu Ner protein. Biochemistry. 1995 Mar 7;34(9):2946–2955. doi: 10.1021/bi00009a026. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Suzuki M., Yagi N. Stereochemical basis of DNA bending by transcription factors. Nucleic Acids Res. 1995 Jun 25;23(12):2083–2091. doi: 10.1093/nar/23.12.2083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taylor J. D., Halford S. E. Discrimination between DNA sequences by the EcoRV restriction endonuclease. Biochemistry. 1989 Jul 25;28(15):6198–6207. doi: 10.1021/bi00441a011. [DOI] [PubMed] [Google Scholar]
  44. Thompson J. F., Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. doi: 10.1093/nar/16.20.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Werner M. H., Huth J. R., Gronenborn A. M., Clore G. M. Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex. Cell. 1995 Jun 2;81(5):705–714. doi: 10.1016/0092-8674(95)90532-4. [DOI] [PubMed] [Google Scholar]
  46. White S. W., Appelt K., Wilson K. S., Tanaka I. A protein structural motif that bends DNA. Proteins. 1989;5(4):281–288. doi: 10.1002/prot.340050405. [DOI] [PubMed] [Google Scholar]
  47. Withers B. E., Dunbar J. C. The endonuclease isoschizomers, SmaI and XmaI, bend DNA in opposite orientations. Nucleic Acids Res. 1993 Jun 11;21(11):2571–2577. doi: 10.1093/nar/21.11.2571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  49. Zinkel S. S., Crothers D. M. Catabolite activator protein-induced DNA bending in transcription initiation. J Mol Biol. 1991 May 20;219(2):201–215. doi: 10.1016/0022-2836(91)90562-k. [DOI] [PubMed] [Google Scholar]
  50. Zinkel S. S., Crothers D. M. DNA bend direction by phase sensitive detection. Nature. 1987 Jul 9;328(6126):178–181. doi: 10.1038/328178a0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES