Inhibition of Escherichia coli RecA coprotease activities by DinI

In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions. The initial step, self-cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single-stranded DNA. In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene. dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids. Immunoblotting analysis with anti-LexA antibodies revealed that LexA did not undergo cleavage in dinI-overexpressed cells after UV irradiation. In addition, the RecA-dependent conversion of UmuD to UmuD' (the active form for mutagenesis) was also inhibited in dinI-overexpressed cells. Conversely, a dinI-deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild-type. Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self-cleavage of UmuD.     

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

How AraC interacts specifically with its target DNAs

Previous work indicates that one subunit of the AraC protein dimer binds to a DNA target araI1, of 17 base-pairs. We systematically substituted every base-pair in a synthetic araI1 target with the three possible alternatives and then tested binding of araI1 and of these 51 DNA targets to AraC by quantitative gel shift analysis in the presence of L-arabinose. We found that every substitution of the underlined bases reduces AraC binding tenfold or more: 5' TAGCATTTTTATCCATA 3'. Substitutions at other bases have little or no effect. In the absence of L-arabinose we observed a sixfold reduction of binding of AraC to araI1. We have designated the 5' AGC sequence the A-box and the 5'TCCATA sequence the B-box. We synthesised DNA targets containing either two A or two B-boxes with the natural araI1-I2 spacing. Wild-type AraC binds both targets in the presence of L-arabinose in a gel shift-experiment. In the absence of L-arabinose, AraC binds only to the double B-box. We then tested various AraC mutant proteins in the same way. S208A and H212A bind to the double B-box but not to the double A-box in the presence or absence of L-arabinose. D256A binds to the double A-box, but not to the double B-box, in the presence of L-arabinose but not in its absence. The implications of these results for the mechanism of AraC induction by L-arabinose are discussed.