DNA degradation in minicells of Escherichia coli K-12. II. Effect of recA1 and recB21 mutations on DNA degradation in minicells and detection of exonuclease V activity
The properties of minicell producing mutants of Escherichia coli deficient in genetic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following uv-irradiation in cells, and the state of DNA metabolism in minicells. The REC$sup -$ phenotypes are unaffected by min$sup +$$/$$sup -$ genotypes in whole cells. In contrast to minicells produced by rec$sup +$ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transferred DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TCA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R64-11 or lambdadv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and [$sup 32$P]-labelled linear DNA from bacteriophage T$sub 7$ for substrate, we found that the ATP- dependent exonuclease of the recBC complex (exo V) is present in rec$sup +$ and recA$sup -$ minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC$sup -$ minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products. (auth)
- Research Organization:
- Univ. of Saskatchewan, Saskatoon
- NSA Number:
- NSA-33-003177
- OSTI ID:
- 4153710
- Journal Information:
- Mol. Gen. Genet., v. 138, pp. 179-192, Journal Name: Mol. Gen. Genet., v. 138, pp. 179-192; ISSN MGGEA
- Country of Publication:
- Germany
- Language:
- English
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