Summary
The D protein encoded by plasmid mini-F promotes resolution of plasmid cointegrates or dimers of the F-factor or mini-F. In addition, two rfsF sequences are essential for this site-specific, recA-independent recombination event. The D gene was cloned into an expression vector and the gene product was overproduced in Escherichia coli and purified to homogeneity. The sequence of the N-terminus of the D protein was determined, thus permitting identification of the correct translational start codon in the nucleotide sequence that results in a 29.6 kDa protein. The binding site for the purified D protein is located within the mini-F NcoIHpaI DNA fragment (192 bp). Binding seems to be affected by DNA methylation, since the protein did not bind to DNA isolated from a dam mutant of E. coli. The binding site, which is a region of approximately 28 bp and is located 160 by downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.
Similar content being viewed by others
References
Ansorge W, Sproat BS, Stegemann J, Schwager C (1986) A nonradioactive automated method for DNA sequence determination. J Biochem Biophys Methods 13:315–323
Austin S, Ziese M, Sternberg N (1981) A novel role for site specific recombination in maintenance of bacterial replicons. Cell 25:729–736
Bachmann BJ (1987) Derivations and genotypes of some mutant derivatives of Escherichia coli K-12. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium, cellular and molecular biology. American Society for Microbiology, Washington DC, pp 1190–1219
Benjamin HW, Matzuk MM, Krasnow MA, Cozzarelli NR (1985) Recombination site selection by Tn3 resolvase: Topological tests of a tracking mechanism. Cell 40:147–158
Bernard HU, Remaut E, Hershfield MV, Das KH, Helinski DR, Yanofsky C, Franklin N (1979) Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda p, promoter. Gene 5:59–76
Bex F, Karoui H, Rokeach L, Drèze P, Garcia L, Couturier M (1983) Mini-F encoded proteins: Identification of a new 10.5 kilodalton species. EMBO J 2:1853–1861
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Chang ACY, Cohen SN (1978) Construction and characterization of amplified multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Disqué-Kochem C, Seidel U, Helsberg M, Eichenlaub R (1985) The repeated sequences (incB) preceding the protein E gene of plasmid mini-F are essential for replication. Mol Gen Genet 202:132–135
de Feyter R, WallaceC, Lane D (1989) Autoregulation of the ccd operon in the F plasmid. Mol Gen Genet 218:481–486
Geier GE, Modrich P (1979) Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of DpnI endonuclease. J Biol Chem 254:1408–1413
Gerlitz M, Hrabak O, Schwab H (1990) Partitioning of broad-hostrange plasmid RP4 is a complex system involving site-specific recombination. J Bacteriol 172:6194–6203
Grindley NDF, Lauth MR, Wells RG, Wityk RJ, Salvo JJ, Reed RR (1982) Transposon — mediated site-specific recombination: identification of three binding sites for resolvase at the res sites of γδ and Tn3. Cell 30:19–27
Hoess RH, Abremski K (1984) Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP. Proc Natl Acad Sci USA 81:1026–1029
Hoess RH, Abremski K (1985) Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J Mol Biel 181:351–362
Kahmann R, Rudt F, Koch C, Mertens G (1985) G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor. Cell 41:771–780
Kanaar R, van de Putte P, Cozzarelli NR (1986) Purification of the Gin recombination protein of Escherichia coli phage Mu and its host factor. Biochim Biophys Acta 866:170–177
Kennedy M, Chandler M, Lane D (1988) Mapping and regulation of the pifC promoter of the F plasmid. Biochim Biophys Acta 950:75–80
Kilbane JJ, Malamy MH (1980) F factor mobilization of nonconjugative chimeric plasmids in Escherichia coli: General mechanisms and a role for site-specific recA-independent recombination at oriV1. J Mol Biol 143:73–93
Komai N, Nishizawa T, Hayakawa Y, Murotsu T, Matsubara K (1982) Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected mini-F segments. Mol Gen Genet 186:193–203
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lane D, de Feyter R, Kennedy M, Phua S-H, Semen D (1986) D protein of miniF plasmid acts as a repressor of transcription and as a site-specific resolvase. Nucleic Acids Res 24:9713–9728
Lovett MA, Helinski DR (1976) Method for the isolation of the replication region of a bacterial replicon: Construction of a mini-F′km plasmid. J Bacteriol 127:982–987
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York pp 250–251
Marinus MG, Morris NR (1974) Biological function for 6-methyladenine residues in the DNA of Escherichia coli K12. J Mol Biol 85:309–322
Mertens G, Klippel A, Fuss H, Blocker H, Frank R, Kahmann R (1988) Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin. EMBO J 7:1219–1227
Miller HI, Friedman DI (1980) An E. coli gene product required for λ site-specific recombination. Cell 20:711–719
Mizuuchi K, Weisberg R, Enquist L, Mizuuchi M, Buraczynska M, Fueller C, Hsu P-L, Ross W, Landy A (1981) Structure and function of the phage γ att site: size, int-binding sites, and location of the crossover point. Cold Spring Harbor Symp Quant Biol 45:429–437
O'Connor MB, Malamy MH (1984a) Role of the F factor oriV1 region in recA-independent illegitimate recombination. Stable replicon fusions of the F derivative pOX38 and pBR322-related plasmids. J Mol Biol 175:263–284
O'Connor MB, Malamy MH (1984b) Site-specific recombination in the oraV1 region of the F factor. Cold Spring Harbor Symp Quant Biol 49:421–434
O'Connor MB, Kilbane JJ, Malamy MH (1986) Site-specific and illegitimate recombination in the oriV1 region of the F factor. DNA sequences involved in recombination and resolution. J Mol Biol 189:85–102
Ogura T, Hiraga S (1983) Mini-F plasmid genes that couple host cell division to plasmid proliferation. Proc Natl Acad Sci USA 80:4784–4788
Remaut E, Stanssens P, Fiers W (1981) Plasmid vectors for high efficiency expression by the p1 promoter of coliphage lambda. Gene 15:81–93
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Tanimoto K, Iino T (1984) An essential gene for replication of the mini-F plasmid from origin I. Mol Gen Genet 196:59–63
Tanimoto K, Iino T (1985) Additional genes essential for replication of the mini-F plasmid from origin I. Mol Gen Genet 198:358–359
Vieira J, Messing J (1982) The pUC plasmids, an M13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Wehlmann H, Eichenlaub R (1980) Plasmid mini-F encoded proteins. Mol Gen Genet 180:205–211
Weisberg RA, Landy A (1983) Site-specific recombination of phage lambda, In: Hendrix R, Roberts J, Stahl F, Weisberg R (eds) Lambda II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York pp 211–250
Woodcock DM, Crother PJ, Doherty J, Jefferson S, DeCruz E, Noyer-Weichner M, Smith SS, Michael MZ, Graham MW (1989) Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17:3469–3478
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Disqué-Kochem, C., Eichenlaub, R. Purification and DNA binding of the D protein, a putative resolvase of the F-factor of Escherichia coli . Molec. Gen. Genet. 237, 206–214 (1993). https://doi.org/10.1007/BF00282802
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00282802