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. 1983 Dec;80(23):7274–7278. doi: 10.1073/pnas.80.23.7274

Directional transport and integration of donor DNA in Haemophilus influenzae transformation.

F Barany, M E Kahn, H O Smith
PMCID: PMC390037  PMID: 6316352

Abstract

DNA transport and integration in Haemophilus influenzae transformation was studied with a plasmid clone of homologous DNA (pCML6). Our results indicate that: (i) donor DNA enters specialized membranous extensions on the cell surface, which we have termed "transformasomes"; (ii) linear DNA undergoes degradation upon exiting transformasomes; and (iii) DNA without a free end remains within transformasomes and is not degraded. By comparing the fate of label from uniformly labeled versus middle-labeled DNA, it appears that donor DNA undergoes degradation from an end prior to recombining with the chromosome. Using donor DNA with covalently closed termini (hairpin ends) prevents efficient exit from transformasomes. When one hairpin is removed, exit of donor DNA is shown to be directional from the free end, with preferential homologous integration of the 3' strand from that end.

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Selected References

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

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Danner D. B., Smith H. O., Narang S. A. Construction of DNA recognition sites active in Haemophilus transformation. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2393–2397. doi: 10.1073/pnas.79.7.2393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. FOX M. S., HOTCHKISS R. D. Fate of transforming deoxyribonucleate following fixation by transformable bacteria. Nature. 1960 Sep 17;187:1002–1006. doi: 10.1038/1871002a0. [DOI] [PubMed] [Google Scholar]
  4. Friedman E. A., Smith H. O. An adenosine triphosphate-dependent deoxyribonuclease from Hemophilus influenzae Rd. 3. Substrate specificity. J Biol Chem. 1972 May 10;247(9):2859–2865. [PubMed] [Google Scholar]
  5. Goodgal S. H. DNA uptake in Haemophilus transformation. Annu Rev Genet. 1982;16:169–192. doi: 10.1146/annurev.ge.16.120182.001125. [DOI] [PubMed] [Google Scholar]
  6. Goodgal S. H., Postel E. H. On the mechanism of integration following transformation with single-stranded DNA of Hemophilus influenzae. J Mol Biol. 1967 Sep 14;28(2):261–273. doi: 10.1016/s0022-2836(67)80008-1. [DOI] [PubMed] [Google Scholar]
  7. Herriott R. M., Meyer E. M., Vogt M. Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J Bacteriol. 1970 Feb;101(2):517–524. doi: 10.1128/jb.101.2.517-524.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kahn M. E., Barany F., Smith H. O. Transformasomes: specialized membranous structures that protect DNA during Haemophilus transformation. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6927–6931. doi: 10.1073/pnas.80.22.6927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kahn M. E., Maul G., Goodgal S. H. Possible mechanism for donor DNA binding and transport in Haemophilus. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6370–6374. doi: 10.1073/pnas.79.20.6370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lacks S., Greenberg B. Competence for deoxyribonucleic acid uptake and deoxyribonuclease action external to cells in the genetic transformation of Diplococcus pneumoniae. J Bacteriol. 1973 Apr;114(1):152–163. doi: 10.1128/jb.114.1.152-163.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lacks S., Greenberg B., Neuberger M. Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2305–2309. doi: 10.1073/pnas.71.6.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lautenberger J. A., Edgell M. H., Hutchison C. A., 3rd The nucleotide sequence recognized by the BstEII restriction endonuclease. Gene. 1980 Dec;12(1-2):171–174. doi: 10.1016/0378-1119(80)90029-3. [DOI] [PubMed] [Google Scholar]
  13. Notani N. K., Setlow J. K. Mechanism of bacterial transformation and transfection. Prog Nucleic Acid Res Mol Biol. 1974;14(0):39–100. doi: 10.1016/s0079-6603(08)60205-6. [DOI] [PubMed] [Google Scholar]
  14. Notani N., Goodgal S. H. On the nature of recombinants formed during transformation in Hemophilus influenzae. J Gen Physiol. 1966 Jul;49(6):197–209. doi: 10.1085/jgp.49.6.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Orlosky M., Smith H. O. Action of ATP-dependent DNase from Hemophilus influenzae on cross-linked DNA molecules. J Biol Chem. 1976 Oct 10;251(19):6117–6121. [PubMed] [Google Scholar]
  16. Radloff R., Bauer W., Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. doi: 10.1073/pnas.57.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Smith G. E., Summers M. D. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem. 1980 Nov 15;109(1):123–129. doi: 10.1016/0003-2697(80)90019-6. [DOI] [PubMed] [Google Scholar]
  18. Smith H. O., Danner D. B., Deich R. A. Genetic transformation. Annu Rev Biochem. 1981;50:41–68. doi: 10.1146/annurev.bi.50.070181.000353. [DOI] [PubMed] [Google Scholar]
  19. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  20. Stuy J. H. Acid-soluble breakdown of homologous deoxyribbonucleic acid adsorbed by Haemophilus influenzae: its biological significance. J Bacteriol. 1974 Nov;120(2):917–922. doi: 10.1128/jb.120.2.917-922.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stuy J. H. Fate of transforming DNA in the Haemophilus influenzae transformation system. J Mol Biol. 1965 Sep;13(2):554–570. doi: 10.1016/s0022-2836(65)80117-6. [DOI] [PubMed] [Google Scholar]
  22. VOLL M. J., GOODGAL S. H. Recombination during transformation in Hemophilus influenzae. Proc Natl Acad Sci U S A. 1961 Apr 15;47:505–512. doi: 10.1073/pnas.47.4.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wilcox K. W., Smith H. O. Binding of the ATP-dependent DNase from Hemophilus influenzae to duplex DNA molecules. J Biol Chem. 1976 Oct 10;251(19):6122–6126. [PubMed] [Google Scholar]
  24. Wilcox K. W., Smith H. O. Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5'-triphosphate-dependent deoxyribonuclease activity. J Bacteriol. 1975 May;122(2):443–453. doi: 10.1128/jb.122.2.443-453.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wilcox K. W., Smith H. O. Mechanism of DNA degradation by the ATP-dependent DNase from Hemophilus influenzae Rd. J Biol Chem. 1976 Oct 10;251(19):6127–6134. [PubMed] [Google Scholar]

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