recC Mutants Research Articles

GENETIC ANALYSIS OF MUTATIONS INDIRECTLY SUPPRESSING recB AND recC MUTATIONS

Mutations in sbcB inactivate exonuclease I and suppress the UV-sensitive, mitomycin-sensitive, recombination-deficient phenotypes associated with recB and recC mutations. Mapping experiments have located sbcB about 0.4 minutes from the his operon at 38.0 on the standard map of E. coli. This places sbcB between supD and his. A four-point cross shows that sbcB lies between P2 attH and his. P2 eduction deleting the his operon beginning with P2 attH also deletes sbcB and produces the expected exonuclease I deficiency and suppression of recB(-). The occurrence of chemical-mutagen-induced and spontaneous mutations indirectly suppressing recB(-) and recC(-) is examined. Three lines of strains produce only sbcA mutations while only sbcB mutations occur in a fourth line. Explanations for this behavior are proposed in light of the ability of the first three lines to express sbcB mutations which they inherit by transduction.

Structural genes of ATP-dependent deoxyribonuclease of Escherichia coli.

RECOMBINATION-deficient (Rec−) mutants of E. coli express pleiotropic alterations of various phenotypes such as increased ultraviolet light sensitivity, altered patterns of DNA degradation after irradiation, inability to support growth of certain λ phage mutants and many others in addition to reduced recipient ability in mating with Hfr bacteria1. Yet the primary function of any one of the genes responsible for these alterations has not been elucidated. In this paper, the characteristics of recB and recC mutants having temperature-sensitive functions are described. Particular attention is paid to the properties of the ATP-dependent deoxyribonuclease which is known to be missing in recB and recC mutants2–5.

Effects of the rec and exr mutations of Escherichia coli on UV reactivation of bacteriophage lambda damaged by different agents

• We confirm that UV reactivation of UV-damaged phage λ does not occur in recA or exr mutants of Escherichia coli. Irradiation of recA and exr bacteria also reduces their ability to perform host-cell reactivation to a much greater extent than wild-type bacteria. RecB and recC mutations have no effect, neither have the phage mutations int and red. • When phage λ is damaged by HNO 2, NH 2OH or nitrogen mustard, UV reactivation occurs in recA and exr mutants. UV-irradiated phage λ in which 5-bromouracil (5-BU) has been incroporated is UV-reactivated in recA but not in exr bacteria. • The breakdown of 32P-labelled phage λ has been followed under different combinations of host and phage irradiation for the different host bacteria. Irradiation of wild-type bacteria depressed the breakdown of irradiated phage. In contrast, breakdown of irradiated or unirradiated phage was increased by irradiation of recA or exr host bacteria. This increase may be analogous to the uncontrolled breakdown of bacterial DNA which occurs after irradiation of recA bacteria. Breakdown itself is not responsible for the elimination of UV reactivation in recA bacteria since the recABC mutant did not show excessive breakdown but has recA phenotype in UV reactivation. • Phage mutagenesis, which normally accompanies UV reactivation, did not occur in recA or exr bacteria although HNO 2-damaged phage was UV-reactivated in the mutant hosts. Clear plaque mutagenesis is not an essential feature of UV reactivation but is linked with recA and exr gene activity. • It is concluded that the recA and exr gene products are not directly involved in UV reactivation, but may be necessary to stabilise or modify UV lesions in phage DNA before reactivation can take place. We have not shown any connection between this activity and clear plaque mutagenesis. Absence of recA or exr gene activity renders UV lesions unrepairable by UV reactivation and, to some extent, by host-cell reactivation. Breakdown of the DNA follows. The different results obtained with different kinds of damage must reflect specificity of the recA and exr gene products for particular types of lesion.

Repair of radiation-induced damage in Escherichia coli: I. Effect of rec mutations on post-replication repair of damage due to ultraviolet radiation

The rec mutants of Escherichia coli K12 are more sensitive to ultraviolet radiation than corresponding rec + derivatives. We have tested several of these rec mutants for post-replication repair after irradiation with ultraviolet light. The mutants carried recA13, recA56, recB21 and recC22. Cultures growing exponentially were exposed to ultraviolet light (63 ergs/mm 2) and then labeled for ten minutes with [ 3H]thymidine. The radioactive medium was removed and the culture divided into two portions, one of which was chilled while the other was reincubated in non-radioactive medium for various times. The DNA from the cells was then analysed on alkaline sucrose gradients. Labeled DNA from irradiated cells which had not been reincubated sedimented as short molecules. After reincubation, DNA from the recA mutants still behaved as short molecules, while DNA from rec +, recB and recC cells assumed a position similar to that of DNA from unirradiated control cells. These results indicate that the two recA mutants tested were defective in their capacity for post-replication of damage induced by ultraviolet radiation. However, no deficiency in the recB and recC mutants was detected under the conditions used. Even in uvr + cells, which are capable of excision-dependent repair, the formation of short molecules could be demonstrated immediately after irradiation. However, after a 40-minute post-irradiation incubation before labeling with [ 3H]thymidine, only “normal-sized” DNA was formed. In contrast, in a uvr − strain, short molecules were still formed after a 90 minute post-irradiation incubation. These results indicate that excision-dependent repair can remove the lesions that lead to the synthesis of short pieces of DNA after ultraviolet-irradiation. Similarly, the exposure of ultraviolet cells to conditions of photoreactivation (which repairs pyrimidine dimers in situ) before labeling greatly diminished but did not completely eliminate the synthesis of short pieces of DNA.

The mutability toward ultarviolet light of recombination-deficient strains of Escherichia coli

Strains having recA and recC mutations were tested for UV mutability. A recA strain is UV-stsable at doses that are demonstrably mutagenic in its Rec+ derivative. Strains of recC genotype are not UV-stable, but produce UV-induced mutations at frequencies significantly lower than those produced at the same doses in Rec+ strains. The results support the hypothesis that most UV-induced mutations originate as errors in recombinational repair of single-strand gaps in DNA.