Repair of the plasmid pBR322 damaged by γ-irradiation or by restriction endonucleases using different recombination-proficient E. coli strains

Abstract

The plasmid pBR322 was treated with BamHI, PvuII and γ-irradiation to generate double-strand breaks (dsb) containing differently structure ends. Transformation efficiencies, mutation frequencies and clone analyses of enzymatically damaged DNA are compared with the corresponding results from radiolytically damaged DNA. In E. coli K-12 SFX, the yield of transformants produced by the action of BamHI, PvuII and γ-irradiation (30 Gy) is 4.3%, 0.14%, and 0.10%, respectively. The survival of open circular DNA (ocDNA) produced by 30 Gy is 1.3%. The transformation efficiencies show only a slight dependence on SOS induction and on the RecA protein. Mutation frequencies to tetracycline sensitivity (tet^S) per surviving plasmid are 2.6% (BamHI), (PvuII) and 0.2% (γ-irradiated DNA with 30 Gy containing ∼ 50% oc DNA and 50% linearized (lin) DNA). The mutation frequency is low at all radiation doses studied (1–50 Gy). Only 15% of the DNA of the tet^S mutants from γ-irradiated plasmids contained deletions whereas with enzymatically damaged DNA, 30–50% (BamHI) or 90% (PvuII) contained deletions. In all cases, all deletions comprised 500–1700 base pairs (bp). After SOS induction of the host cells, the mutation frequency of γ-irradiated plasmids increased by a factor of 4, whereas that of the enzymatically damaged plasmids did not change.

For the repair of the enzymatically linearized DNA 2 recombinational pathways are discussed which lead to deletant (pathway I) and non-deletant transformants (pathway II). In addition, BamHI-linearized plasmids may be repaired by enzyme-induced or spontaneous circular alignment followed by ligation. The high percentage of deletions of the tet^S mutations for PvuII-linearized DNA with the blunt ends is explained by the illegitimate or site-specific recombination pathway I (see text). The lower percentage of deletions of the tet^S mutations with BamHI-linearized DNA with short cohesive ends (4 bp) is proposed to be due to a greater contribution of pathway II and/or by circular alignment followed by ligation. The very small yield and the low percentage of deletant mutations of tet^S mutants from radiolytically damaged DNA is proposed to be due to the large overlapping ends (16–100 bp) of the linDNA which easily leads to circular alignment followed by excision repair. The repair of radiolytically produced ocDNA is predominantly due to excision repair. In agrement with this interpretation is the observation that SOS induction of the host increases the mutation incidence of radiolytically damaged DNA but not of enzymatically damaged DNA.