Richard P. Boyce

DNA REPAIR AND GENETIC RECOMBINATION: STUDIES ON MUTANTS OF ESCHERICHIA COLI DEFECTIVE IN THESE PROCESSES.

A theory for the mechanism of enzymic repair of DNA containing ultraviolet (UV) photoproducts in UV-irradiated bacteria was proposed in 1964 (1, 2). Subsequent experiments have lent further support to this theory and have provided evidence that it may be applicable to the enzymic repair of products formed in DNA by a number of different mutagens, including alkylating agents and X-rays. It has also been shown that DNA repair of a similar kind may play an essential role in genetic recombination. With these latter findings taken into consideration, the following more comprehensive theory for the mechanism and functions of DNA repair can be formulated. Four main steps are involved in DNA repair: 1. Single strands of two-strand DNA are interrupted in either of several ways; (a) by a radiation-induced chain break; (b) by the enzymic excision of damaged bases; or (c) by a recombination enzyme that acts during the early stages of genetic recombination. 2. Nucleotides are released, presumably through the action of an enzyme on the free single-strand ends left by these cuts. This enzyme appears to proceed only for a limited distance in normal cells. 3. The DNA twin helix is reconstructed by a DNA repair polymerase that inserts complementary nucleotides into the gap, adding them onto one of the singlestrand ends. Thus, the end of a single strand serves as initiator, while the intact strand opposite serves as template. 4. The repair is completed by joining the phosphodiester backbone when the last nucleotide is inserted into the gap. The mode of action of this repair mechanism in removing a thymine dimer from DNA is illustrated in Fig. 1. The first section of this paper will review experiments on the mechanisms of DNA repair in Escherichia coli following exposure to irradiation or to mutagens. In the next section the role of DNA repair in genetic recombination will be dis-

Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli K12.

A given dose of ultraviolet irradiation produces as many pyrimidine-dimer-containing photoproducts in the DNA of the Escherichia coli Rec − strain JC1569 as it does in the related Rec + strain JC1557. Both strains excise dimer-containing photoproducts from their DNA to the same extent. In comparison with JC1557, approximately 30 times as much DNA of the Rec − strain is degraded per unit dose of ultraviolet light. Perhaps as a result of this excess ultraviolet-induced degradation, irradiated Rec − cells do not incorporate appreciable amounts of exogenous thymidine or thymine into DNA and have few survivors following exposure to an ultraviolet dose which almost all Rec + cells survive. A numerical argument is offered to establish the ability of Rec − cells successfully to replace excised pyrimidine-dimer-containing regions of the DNA with normal nucleotide residues. No effect of the rec − mutation in JC1569 was found on the level of activity of the following enzymes: endonuclease I, exonuclease I, exonuclease III and DNA phosphatase, and DNA polymerase and exonuclease II.

Genetic control of DNA breakdown and repair inE. coli K-12 treated with mitomycin C or ultraviolet light

SummaryUltraviolet light sensitive mutants ofE. coli defective at theuvrA,uvrB oruvrC locus showed increased sensitivity to the lethal effects of mitomycin C when compared with theuvr+ parental strain. In addition, DNA breakdown after treatment of cells with either mitomycin C or with ultraviolet light was greater in the parental strain carrying the activeuvr+ genes than inuvr mutants. Thus, injuries produced by either mitomycin C or by ultraviolet light may be repaired by the same molecular mechanism which has been proposed and which involves defect excision, single strand breakdown and reconstruction of the DNA.

Production of single-strand breaks in covalent circular λ phage DNA in superinfected lysogens by monoalkylating agents and the joining of broken DNA strands

Fast sedimenting, double-stranded, covalent circular lambda phage DNA in superinfected lysogens of Escherichia coli is converted to notched circular DNA, sedimenting slowly in alkaline sucrose gradients, by methylmethane sulfonate (MMS) treatment during incubation at 37 degrees . During further incubation after removal of the MMS, the slow sedimenting DNA reverts to the fast sedimenting structure. Covalent circular DNA, phenol extracted from superinfected lysogens and exposed to MMS, ethylmethane sulfonate, or 2-chloroethyl ethyl sulfide requires the presence of a heat-labile factor from extracts of E. coli for the breaks to appear. The cleavage of alkylated covalent circular DNA in vivo and the subsequent rejoining of the broken DNA strands to produce reconstructed covalent circular DNA may reflect the action of an excision-like repair process which is under different genetic control from that of pyrimidine dimer excision.

X-RAY-INDUCED SINGLE-STRAND BREAKS AND JOINING OF BROKEN STRANDS IN SUPERINFECTING lambda DNA IN ESCHERICHIA COLI LYSOGENIC FOR lambda.

Abstract Covalently bonded circular λ phage DNA in superinfected lysogens of Escherichia coli is converted to notched circular DNA by X-irradiation at 0°. The kinetics of conversion are first order with an 1 e dose of 22 krads in the presence of O2 and 62 krads in the presence of N2. Greater than 50% of the notched circular DNA produced by irradiation of the superinfected cells reverts to covalently bonded circular DNA within the first 5 minutes upon incubation at 37°. The conversion of notched circular DNA to covalently bonded DNA occurs normally in various radiation-sensitive mutants of E. coli, including those deficient in recombination.

The Influence of the λ Prophage on the Action of the Inducible Inhibitor of Postirradiation DNA Degradation@@@The Influence of the l Prophage on the Action of the Inducible Inhibitor of Postirradiation DNA Degradation

Early observations on the inducible inhibitor of postirradiation DNA degradation involved cells of Eschericia coli which carried either prophage or defective prophage. The question as to whether the prophage element is essential can be answered by observing the induction of inhibition in cells which (a) carry the prophage and can be induced or do not have the prophage and (b) carry the prophage but can not be induced. The strains used are AB1157 and AB1157

RELEASE OF ULTRAVIOLET LIGHT-INDUCED THYMINE DIMERS FROM DNA IN E. COLI K-12

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Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA.

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A simple method of increasing the incorporation of thymidine into the deoxyribonucleic acid of Escherichia coli

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Mechanism of sensitization to ultra-violet light of T1 bacteriophage by the incorporation of 5-bromodeoxyuridine or by pre-irradiation of the host cell.

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