Mutational Analysis of a Prokaryotic Recombinational Enhancer Element with Two Functions

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1989 Feb 1

PMID 2656257

Citations 61

Authors

P Hubner

W Arber

Affiliations

Soon will be listed here.

Abstract

The site-specific DNA inversion system Cin encoded by the bacteriophage P1 consists of a recombinase, two inverted crossing-over sites and a recombinational enhancer. The latter approximately 75 bp long genetic element is bifunctional due to its location within the 5' part of the cin gene encoding the recombinase. In order to determine the essential nucleotides for each of its two biological functions we randomly mutated the recombinational enhancer sequence sis(P1) and analysed both functions of the mutants obtained. Three distinct regions of this sequence were found to be important for the enhancer activity. One of them occupies the middle third of the enhancer sequence and it can suffer a number of functionally neutral base substitutions, while others are detrimental. The other two regions occupy the two flanking thirds of the enhancer. They coincide with binding sites of the host-coded protein FIS (Factor for Inversion Stimulation) needed for efficient DNA inversion in vitro. These sequences appear to be highly evolved allowing only a few mutations without affecting either of the biological functions. Taking the effect of mutations within these FIS binding sites into account a consensus sequence for the interaction with FIS was compiled. This FIS consensus implies a palindromic structure for the recombinational enhancer. This is in line with the orientation independence of enhancer action with respect to the crossing-over sites.

Citing Articles

DNA Sequence Determinants Controlling Affinity, Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis.

Hancock S, Stella S, Cascio D, Johnson R PLoS One. 2016; 11(3):e0150189.

PMID: 26959646 PMC: 4784862. DOI: 10.1371/journal.pone.0150189.

Site-specific DNA Inversion by Serine Recombinases.

Johnson R Microbiol Spectr. 2015; 3(3):1-36.

PMID: 25844275 PMC: 4384473. DOI: 10.1128/microbiolspec.MDNA3-0047-2014.

Atomic force microscopy analysis of the role of major DNA-binding proteins in organization of the nucleoid in Escherichia coli.

Ohniwa R, Muchaku H, Saito S, Wada C, Morikawa K PLoS One. 2013; 8(8):e72954.

PMID: 23951337 PMC: 3741201. DOI: 10.1371/journal.pone.0072954.

Silencing of toxic gene expression by Fis.

Karambelkar S, Swapna G, Nagaraja V Nucleic Acids Res. 2012; 40(10):4358-67.

PMID: 22287621 PMC: 3378877. DOI: 10.1093/nar/gks037.

The shape of the DNA minor groove directs binding by the DNA-bending protein Fis.

Stella S, Cascio D, Johnson R Genes Dev. 2010; 24(8):814-26.

PMID: 20395367 PMC: 2854395. DOI: 10.1101/gad.1900610.

References

Plasterk R, Van de Putte P . Genetic switches by DNA inversions in prokaryotes. Biochim Biophys Acta. 1984; 782(2):111-9. DOI: 10.1016/0167-4781(84)90013-7. View

Huber H, Iida S, Arber W, Bickle T . Site-specific DNA inversion is enhanced by a DNA sequence element in cis. Proc Natl Acad Sci U S A. 1985; 82(11):3776-80. PMC: 397870. DOI: 10.1073/pnas.82.11.3776. View

Iida S . Sequence of the site-specific recombinase gene cin and of its substrates serving in the inversion of the C segment of bacteriophage P1. EMBO J. 1983; 2(10):1733-40. PMC: 555351. DOI: 10.1002/j.1460-2075.1983.tb01650.x. View

Schmucker R, Ritthaler W, Stern B, Kamp D . DNA inversion in bacteriophage Mu: characterization of the inversion site. J Gen Virol. 1986; 67 ( Pt 6):1123-33. DOI: 10.1099/0022-1317-67-6-1123. View

Klippel A, Mertens G, Patschinsky T, Kahmann R . The DNA invertase Gin of phage Mu: formation of a covalent complex with DNA via a phosphoserine at amino acid position 9. EMBO J. 1988; 7(4):1229-37. PMC: 454460. DOI: 10.1002/j.1460-2075.1988.tb02935.x. View

Sadowski P . Site-specific recombinases: changing partners and doing the twist. J Bacteriol. 1986; 165(2):341-7. PMC: 214421. DOI: 10.1128/jb.165.2.341-347.1986. View

Kamp D, Chow L, Broker T, Kwoh D, Zipser D, Kahmann R . Site-specific recombination in phage mu. Cold Spring Harb Symp Quant Biol. 1979; 43 Pt 2:1159-67. DOI: 10.1101/sqb.1979.043.01.131. View

Kunkel T . Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985; 82(2):488-92. PMC: 397064. DOI: 10.1073/pnas.82.2.488. View

Haffter P, Bickle T . Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin. J Mol Biol. 1987; 198(4):579-87. DOI: 10.1016/0022-2836(87)90201-4. View

10.

SANGER F, Nicklen S, Coulson A . DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977; 74(12):5463-7. PMC: 431765. DOI: 10.1073/pnas.74.12.5463. View

11.

Koch C, Kahmann R . Purification and properties of the Escherichia coli host factor required for inversion of the G segment in bacteriophage Mu. J Biol Chem. 1986; 261(33):15673-8. View

12.

Zieg J, Simon M . Analysis of the nucleotide sequence of an invertible controlling element. Proc Natl Acad Sci U S A. 1980; 77(7):4196-200. PMC: 349798. DOI: 10.1073/pnas.77.7.4196. View

13.

Koch C, Vandekerckhove J, Kahmann R . Escherichia coli host factor for site-specific DNA inversion: cloning and characterization of the fis gene. Proc Natl Acad Sci U S A. 1988; 85(12):4237-41. PMC: 280402. DOI: 10.1073/pnas.85.12.4237. View

14.

Pabo C, Sauer R . Protein-DNA recognition. Annu Rev Biochem. 1984; 53:293-321. DOI: 10.1146/annurev.bi.53.070184.001453. View

15.

Silverman M, Zieg J, Mandel G, Simon M . Analysis of the functional components of the phase variation system. Cold Spring Harb Symp Quant Biol. 1981; 45 Pt 1:17-26. DOI: 10.1101/sqb.1981.045.01.005. View

16.

Johnson R, Ball C, PFEFFER D, Simon M . Isolation of the gene encoding the Hin recombinational enhancer binding protein. Proc Natl Acad Sci U S A. 1988; 85(10):3484-8. PMC: 280236. DOI: 10.1073/pnas.85.10.3484. View

17.

Plasterk R, Brinkman A, Van de Putte P . DNA inversions in the chromosome of Escherichia coli and in bacteriophage Mu: relationship to other site-specific recombination systems. Proc Natl Acad Sci U S A. 1983; 80(17):5355-8. PMC: 384254. DOI: 10.1073/pnas.80.17.5355. View

18.

Iino T, Kutsukake K . Trans-acting genes of bacteriophages P1 and Mu mediate inversion of a specific DNA segment involved in flagellar phase variation of Salmonella. Cold Spring Harb Symp Quant Biol. 1981; 45 Pt 1:11-6. DOI: 10.1101/sqb.1981.045.01.004. View

19.

Iida S . Bacteriophage P1 carries two related sets of genes determining its host range in the invertible C segment of its genome. Virology. 1984; 134(2):421-34. DOI: 10.1016/0042-6822(84)90309-x. View

20.

Johnson R, Glasgow A, Simon M . Spatial relationship of the Fis binding sites for Hin recombinational enhancer activity. Nature. 1987; 329(6138):462-5. DOI: 10.1038/329462a0. View