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. 1996 Sep 2;15(17):4775–4783.

Generation of new DNA binding specificity by truncation of the type IC EcoDXXI hsdS gene.

M P MacWilliams 1, T A Bickle 1
PMCID: PMC452210  PMID: 8887569

Abstract

The hsdS subunit of a type IC restriction-modification enzyme is responsible for the enzyme's DNA binding specificity. Type I recognition sites are characterized by two defined half-sites separated by a non-specific spacer of defined length. The hsdS subunit contains two independent DNA binding domains, each targeted towards one DNA half-site. We have shown previously that the 5' half of hsdS can code for a functional substitute of the full-length hsdS. Here we demonstrate that the 3' half of the gene, when fused to the appropriate transcriptional and translational start signals, also codes for a peptide which imparts DNA binding specificity to the enzyme. About half the natural hsdS size, the mutant peptide contains a single DNA recognition domain flanked by one copy of each internal repeat found in the full-length hsdS. Deletion of either repeat sequence results in loss of activity. Like the 5' hsdS mutant, the 3' mutant recognizes an interrupted palindrome, GAAYN(5)RTTC, suggesting that two truncated subunits participate in DNA recognition. Co-expression of the 5' hsdS mutant and the 3' hsdS mutant along with hsdM regenerates the wild-type methylation specificity. Thus, there is a free assortment of subunits in the cell.

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Selected References

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  1. Abadjieva A., Patel J., Webb M., Zinkevich V., Firman K. A deletion mutant of the type IC restriction endonuclease EcoR1241 expressing a novel DNA specificity. Nucleic Acids Res. 1993 Sep 25;21(19):4435–4443. doi: 10.1093/nar/21.19.4435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abadjieva A., Webb M., Patel J., Zinkevich V., Firman K. Deletions within the DNA recognition subunit of M.EcoR124I that identify a region involved in protein-protein interactions between HsdS and HsdM. J Mol Biol. 1994 Aug 5;241(1):35–43. doi: 10.1006/jmbi.1994.1471. [DOI] [PubMed] [Google Scholar]
  3. Alting-Mees M. A., Short J. M. pBluescript II: gene mapping vectors. Nucleic Acids Res. 1989 Nov 25;17(22):9494–9494. doi: 10.1093/nar/17.22.9494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Argos P. Evidence for a repeating domain in type I restriction enzymes. EMBO J. 1985 May;4(5):1351–1355. doi: 10.1002/j.1460-2075.1985.tb03784.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barcus V. A., Titheradge A. J., Murray N. E. The diversity of alleles at the hsd locus in natural populations of Escherichia coli. Genetics. 1995 Aug;140(4):1187–1197. doi: 10.1093/genetics/140.4.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bickle T. A., Krüger D. H. Biology of DNA restriction. Microbiol Rev. 1993 Jun;57(2):434–450. doi: 10.1128/mr.57.2.434-450.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bullas L. R., Colson C., Van Pel A. DNA restriction and modification systems in Salmonella. SQ, a new system derived by recombination between the SB system of Salmonella typhimurium and the SP system of Salmonella potsdam. J Gen Microbiol. 1976 Jul;95(1):166–172. doi: 10.1099/00221287-95-1-166. [DOI] [PubMed] [Google Scholar]
  8. Chen A., Powell L. M., Dryden D. T., Murray N. E., Brown T. Tyrosine 27 of the specificity polypeptide of EcoKI can be UV crosslinked to a bromodeoxyuridine-substituted DNA target sequence. Nucleic Acids Res. 1995 Apr 11;23(7):1177–1183. doi: 10.1093/nar/23.7.1177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooper L. P., Dryden D. T. The domains of a type I DNA methyltransferase. Interactions and role in recognition of DNA methylation. J Mol Biol. 1994 Mar 4;236(4):1011–1021. doi: 10.1016/0022-2836(94)90008-6. [DOI] [PubMed] [Google Scholar]
  10. Daniel A. S., Fuller-Pace F. V., Legge D. M., Murray N. E. Distribution and diversity of hsd genes in Escherichia coli and other enteric bacteria. J Bacteriol. 1988 Apr;170(4):1775–1782. doi: 10.1128/jb.170.4.1775-1782.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dybvig K., Yu H. Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis. Mol Microbiol. 1994 May;12(4):547–560. doi: 10.1111/j.1365-2958.1994.tb01041.x. [DOI] [PubMed] [Google Scholar]
  12. Fuller-Pace F. V., Bullas L. R., Delius H., Murray N. E. Genetic recombination can generate altered restriction specificity. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6095–6099. doi: 10.1073/pnas.81.19.6095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fuller-Pace F. V., Murray N. E. Two DNA recognition domains of the specificity polypeptides of a family of type I restriction enzymes. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9368–9372. doi: 10.1073/pnas.83.24.9368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gann A. A., Campbell A. J., Collins J. F., Coulson A. F., Murray N. E. Reassortment of DNA recognition domains and the evolution of new specificities. Mol Microbiol. 1987 Jul;1(1):13–22. doi: 10.1111/j.1365-2958.1987.tb00521.x. [DOI] [PubMed] [Google Scholar]
  15. Gubler M., Bickle T. A. Increased protein flexibility leads to promiscuous protein--DNA interactions in type IC restriction-modification systems. EMBO J. 1991 Apr;10(4):951–957. doi: 10.1002/j.1460-2075.1991.tb08029.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gubler M., Braguglia D., Meyer J., Piekarowicz A., Bickle T. A. Recombination of constant and variable modules alters DNA sequence recognition by type IC restriction-modification enzymes. EMBO J. 1992 Jan;11(1):233–240. doi: 10.1002/j.1460-2075.1992.tb05046.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. JACOB F., WOLLMAN E. L. Etude génétique d'un bactériophage tempéré d'Escherichia coli. l. Le système genétique du bactériophage. Ann Inst Pasteur (Paris) 1954 Dec;87(6):653–673. [PubMed] [Google Scholar]
  18. Kannan P., Cowan G. M., Daniel A. S., Gann A. A., Murray N. E. Conservation of organization in the specificity polypeptides of two families of type I restriction enzymes. J Mol Biol. 1989 Oct 5;209(3):335–344. doi: 10.1016/0022-2836(89)90001-6. [DOI] [PubMed] [Google Scholar]
  19. Meister J., MacWilliams M., Hübner P., Jütte H., Skrzypek E., Piekarowicz A., Bickle T. A. Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition. EMBO J. 1993 Dec;12(12):4585–4591. doi: 10.1002/j.1460-2075.1993.tb06147.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nagaraja V., Shepherd J. C., Bickle T. A. A hybrid recognition sequence in a recombinant restriction enzyme and the evolution of DNA sequence specificity. Nature. 1985 Jul 25;316(6026):371–372. doi: 10.1038/316371a0. [DOI] [PubMed] [Google Scholar]
  21. Price C., Lingner J., Bickle T. A., Firman K., Glover S. W. Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes. J Mol Biol. 1989 Jan 5;205(1):115–125. doi: 10.1016/0022-2836(89)90369-0. [DOI] [PubMed] [Google Scholar]
  22. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tyndall C., Meister J., Bickle T. A. The Escherichia coli prr region encodes a functional type IC DNA restriction system closely integrated with an anticodon nuclease gene. J Mol Biol. 1994 Apr 1;237(3):266–274. doi: 10.1006/jmbi.1994.1230. [DOI] [PubMed] [Google Scholar]
  24. Valinluck B., Lee N. S., Ryu J. A new restriction-modification system, KpnBI, recognized in Klebsiella pneumoniae. Gene. 1995 Dec 29;167(1-2):59–62. doi: 10.1016/0378-1119(95)00660-5. [DOI] [PubMed] [Google Scholar]
  25. Wilson G. G., Murray N. E. Restriction and modification systems. Annu Rev Genet. 1991;25:585–627. doi: 10.1146/annurev.ge.25.120191.003101. [DOI] [PubMed] [Google Scholar]
  26. Wood W. B. Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol. 1966 Mar;16(1):118–133. doi: 10.1016/s0022-2836(66)80267-x. [DOI] [PubMed] [Google Scholar]
  27. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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