Erling Seeberg

Cloning of Escherichia coli genes encoding 3-methyladenine DNA glycosylases I and II

SummaryWe have constructed two recombinant plasmids which harbour functions involved in DNA repair of alkylation damage in Escherichia coli. One plasmid carries the tag+ gene encoding 3-methyladenine DNA glycosylase I while the other carries alkA+ encoding 3-methyladenine DNA glycosylase II. The plasmids were isolated from plasmid stocks carrying total cellular DNA by selection for their ability to complement the methylmethanesulphonate(MMS)-sensitive phenotype of an E. coli mutant (tag ada) deficient in both 3-methyladenine DNA glycosylases I and II. Both plasmids increase the plating efficiency of such a mutant on methylmethanesulphonate plates by a factor of more than 105. The tag gene is located on a 6 (kbp) HindIII fragment, and the presence of the tag plasmid in the cells results in 15-fold overproduction of 3-methyladenine DNA glycosylase I. The other plasmid restores 3-methyladenine DNA glycosylase II deficiency in alkA mutant cells, and results in 3-fold overproduction of this enzyme after alkylation induction. The induction is ada+-dependent and we conclude that this plasmid contains the structural gene for 3-methyladenine DNA glycosylase II, including its control region responding to alkylation induction. However, the plasmid does not complement fully the MMS-sensitive phenotype of alkA mutants which suggests that the plasmid may not include the entire alkA operon.

Incisions in ultraviolet irradiated circular bacteriophage lambda DNA molecules in excision proficient and deficient lysogens of E. coli.

SummaryThe breakage of closed covalent λ DNA molecules in lysogenic host cells after ultravilet irradiation was investigated. In repair proficient host cells incisions are introduced immediately following irradiation. A steady-state of strand interruptions is observed within 20–50 seconds after irradiation, where the number of broken molecules is dose dependent for doses up to 600 ergs/mm2. No ultraviolet promoted strand breaks were observed in uvrA or uvrB lysogens, in accordance with previous results obtained by Shimada, Ogawa & Tomizawa [Molec. gen. Genet. 101, 245 (1968)]. In contrast, uvrC mutants have the ability to form breaks in superinfecting λ DNA molecules after ultraviolet irradiation. The ultraviolet specific endonucleolytic activity observed in uvrC host cells differs from that observed in uvr+ host cells in that, (i) the first break is introduced at least 15 times slower, (ii) for doses below 300 ergs/mm2 the number of strand breaks is higher, (iii) the dose dependence terminates at a lower dose. The possible function of the uvrC gene product in the repair is discussed.

Strand cleavage at psoralen adducts and pyrimidine dimers in DNA caused by interaction between semi-purified uvr+ gene products from Escherichia coli

Partially purified extracts of Escherichia coli containing either uvrA+ or a mixture of uvrB+ and uvrC+ gene products were tested for an endonuclease activity on DNA treated with 8-methoxypsoralen plus 360-nm light. Neither of these fractions was active alone. The combined fractions, however, caused extensive strand cleavage of the psoralen-treated DNA. The endonuclease activity was dependent upon addition of ATP and Mg2+ to the reaction mixtures, and hence appeared similar to the UV-endonuclease activity previously shown to be reconstituted from the same fractions. It is concluded that the uvr+ gene products in these fractions interact to cause breakage of both psoralen-treated and UV-irradiated DNA. An examination of the dose-dependence relationship of the break formation in psoralen-treated DNA revealed that the enzyme acts upon psoralen mono-adducts. By varying the experimental conditions to increase the ratio of interstrand cross-links to mono-adducts it was found that the enzyme also acts upon cross-links, but with lower efficiency than for mono-adducts. Further studies of break formation in UV-irradiated DNA showed that elimination of pyrimidine dimers by treatment with photoreactivating enzyme in the light resulted in a loss of endonuclease-sensitive sites. This shows directly that pyrimidine dimers are the lesions recognized by the complemented uvr+ gene products in UV-irradiated DNA. For comparison, another endonuclease acting at pyrimidine dimers in DNA, the Micrococcus luteus UV-endonuclease, was also tested with psoralen-treated DNA, but no activity was observed. This and other data indicate that the repair endonuclease encoded by the uvr+ genes in E. coli is basically different from the other dimer-specific endonucleases previously characterized.

Multiprotein Interactions in Strand Cleavage of DNA Damaged by UV and Chemicals

Publisher Summary The chapter explains the characterization of the products of genes uvrA, uvrB, and uvrC purified by means of the complementation assay. It has long been assumed that the initial step of excision repair of UV-irradiated DNA in E.coli is similar to that characterized in vitro by the small dimer-specific glycosylases from M.luteus and T4-infected cells. This assumption has prevented major efforts to elucidate the uvr+-dependent incision enzyme in E coli . However, recent studies of the uvr+ gene products in vitro show that these proteins are basically different from the small DNA glycosylases characterized so far. The interactions between the uvrA+, uvrB+, and uvrC+ gene products resulting in the appearance of strand breaks in DNA damaged by UV and various chemical agents are now being subjected to extensive studies in several laboratories, and it is to be expected that a more detailed knowledge about the excision mechanism in E.coli .

REPAIR OF PSORALEN PLUS NEAR ULTRAVIOLET LIGHT DAMAGE IN BACTERIOPHAGES T3 AND T4

Abstract— Repair of T3 and T4 DNA damaged by treatment with 8‐methoxypsoralen plus near UV (PNUV) has been investigated. It is shown that the excision repair mechanisms of the host cell can repair a substantial fraction of the psoralen‐DNA mono‐adducts in T3 DNA, but cannot by themselves repair crosslinks. In contrast neither the excision repair system of the host nor the phage coded v gene endonuclease is involved in the repair of psoralen adducts in T4 DNA. Multiplicity reactivation is effective in the recovery of T4 DNA containing psoralen‐DNA mono‐adducts, but is ineffective in the recovery of crosslinked phages. Comparisons of the lethality of PNUV treatment and the number of crosslinks induced in T4 DNA show clearly that mono‐adducts are lethal to this phage. Both T3 and T4, however, appear to effectively repair many mono‐adducts by postreplicational repair.

Cloning of the uvrC + Gene from Escherichia coli Onto a Plaque Forming Phage Vector

Specialized transducing phages are important tools in the characterization of host coded gene functions. Isolation of specialized transducing phages was previously restricted to those carrying host genes located close to the normal phage attachment sitesl. However, recent advances in techniques of DNA recombination both in vivo and in vitro have made possible in principle the isolation of transducing phages carrying genes from anywhere on the bacterial chromosome2–4. This communication describes the isolation of a plaque-forming bacteriophage λ carrying the E.coli uvrC+ gene.

A DNA-BINDING ACTIVITY ASSOCIATED WITH THE uvrA+ PROTEIN FROM Escherichia coli

ABSTRACT An in vitro complementation assay has been used for the partial purification of the uvrA protein from E. coli ( 1 ). The uvrA protein is recovered in a high molecular weight form (MW − 100,000) and has no detectable endonuclease activity, but is associated with a DNA-binding activity with preferential affinity for UV-irradiated DNA. This binding activity is absent from a uvrA mutant strain and therefore is possibly an inherent property of the uvrA protein itself.

Two separable protein species which both restore uvrABC endonuclease activity in extracts from uvrC mutated cells

Two different protein species which both complement the detective repair endonuclease (uvrABC endonuclease) in uvrC mutated cells have been detected. These proteins have quite different chromatographic properties and were easily separated by ion exchange chromatography. One has affinity for DEAE cellulose and co-cromatographs with the uvrB protein. The other has strong affinity for phosphocellulose and appears to be the uvrC protein itself. The uvrB associated uvrC+ activity is absent from both uvrC and uvrB mutated cells, indicating that this species result from an interaction between uvrB+ and uvrC+ functions at the protein level.