Dimerization of plasmid DNA accelerates selection for antibiotic resistance.

Dimerization of multicopy plasmids is widely assumed to be disadvantageous both for plasmid maintenance and for the host cell. It is known that dimerization causes plasmid instability; dimer-containing cells grow slower than their monomer-containing counterparts. However, as we demonstrate here, under conditions of selective stress, dimers provide an advantage for bacteria. Dimers facilitate segregation of mutants from numerous copies of the parental plasmid. Accelerated segregation greatly increases the rate of accumulation of plasmids carrying mutations that are adaptive for bacteria. In contrast, resolution of dimers by site-specific recombination decreases, 10(3)-10(5)-fold, the efficiency of selection of spontaneous reversions in the tet gene of pBR327.

Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break and deletion formation.

The role of closely spaced lesions on both DNA strands in the induction of double-strand breaks and formation of deletions was studied. For this purpose a polylinker sequence flanked by 165 bp direct repeats was inserted within the tet gene of pBR327. This plasmid was used to construct DNA containing one or two uracil residues which replaced cytosine residues in the KpnI restriction site of the polylinker. Incubation of the plasmid DNA construct with Escherichia coli cell-free extracts showed that double-strand breaks occurred as a result of excision repair of the opposing uracil residues by uracil-DNA glycosylase (in extracts from ung+ but not in extracts from ung- E. coli strains). Recombination of direct repeats, induced by double-strand breakage of plasmid DNA, can lead to the deletion of the polylinker and of one of the direct repeats, thus restoring the tet+ gene function which can be detected by the appearance of tetracycline-resistant colonies of transformants. Transformation of E. coli cells with single or double uracil-containing DNAs demonstrated that DNA containing two closely spaced uracil residues was tenfold more effective in the induction of deletions than DNA containing only a single uracil residue. The frequency of deletions is increased tenfold in an ung+ E. coli strain in comparison with an ung- strain, suggesting that deletions are induced by double-strand breakage of plasmid DNA which occurs in vivo as a result of the excision of opposing uracil residues.

[A method for inducing the point C----T transitions in DNA sequences corresponding to the ends of restriction sites]. / Metod polucheniia tochkovykh C----T tranzitsii po uchastkam DNK, sootvetstvuiushchim kontsam saitov restriktsii.

A new method for inducing of C----T substitutions into cytosine-containing restriction sites is developed. The method, based on the selective modification of cytosine residues in DNA sticky ends by sodium bisulfite, was illustrated by induction of a base substitution (C----T at the BamHI site of pBR322 plasmid DNA.