Purification and DNA binding of the D protein, a putative resolvase of the F-factor of Escherichia coli.
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 NcoI-HpaI 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 bp downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.
Disqué-Kochem
C
C
Universität Bielefeld, Fakultät für Biologie, Lehrstuhl für Gentechnologie/Mikrobiologie, Bielefeld, FRG.
Eichenlaub
R
R
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005144:F Factor / Q000378:metabolism*; D008780:Methyltransferases; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D017421:Sequence Analysis; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D019895:Transposases
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D008780:Methyltransferases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499294:dam protein, E coli; D009713:Nucleotidyltransferases; D019895:Transposases