Ultraviolet-B-induced damage to Escherichia coli Fpg protein.

We investigated the effect of UVB light (290 < or = lambda < or = 320 nm) on the structure and enzymatic activities of Escherichia coli Fpg protein (2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine-DNA glycosylase), a DNA repair enzyme containing a zinc finger motif and five chromophoric Trp residues. Irradiation with UVB light of air-saturated pH 7.4 buffered aqueous solutions of Fpg induces the formation of polymers as shown by sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis. In argon-saturated solutions, polymer formation produces a precipitate. The polymerization quantum yield is 0.07 +/- 0.01 and 0.15 +/- 0.02 in air- and argon-saturated solutions, respectively. In the polymerized Fpg protein, second-derivative absorption spectroscopy indicates that three and one Trp residues are destroyed in air- and argon-saturated solutions, respectively. Polymers are devoid of all three activities of the Fpg protein, whereas the unpolymerized protein retains full activities. Matrix-assisted laser desorption/ionization experiments demonstrate that polymer formation is accompanied by the formation of short polypeptides containing the first 32 or 33 residues of the N-terminal domain. Theses polypeptides are most probably formed by the photolytic cleavage of Fpg protein induced by light absorption by the adjacent Trp-34 residue.

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway. I: DNA base and nucleolytic specificity.

Cells of Bacillus subtilis can enter a natural physiological state, termed competence, that is permissive for uptake of DNA from the surrounding medium. In the B. subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, followed by intracellular processing that may ultimately lead to productive infection. Previous investigations have shown that transfecting DNA is usually far less infectious (on a molar basis) than is the DNA injected by phage particles; this result is apparently due to inactivating events suffered by transfecting DNA during its metabolism by competent cells. Earlier studies also demonstrated that, in some cases, the infectivity of transfecting DNA can be increased by ultraviolet (UV) irradiation of the competent cells prior to transfection, or by cotransfection of UV-irradiated heterologous DNAs; collectively, these phenomena have been termed transfection enhancement (TE). We propose here that some transfecting B. subtilis phage DNAs are attacked by a novel host DNA repair system, and that TE reflects inhibition of this by a competing substrate in UV-irradiated DNA. In support of this model, we show that UV-DNA cotransfection leads to a reduced rate of intracellular endonucleolytic breakdown of transfecting DNA. We also demonstrate that TE displays marked specificity of a kind frequently observed for repair enzymes. Thus, phages that contain hydroxymethyl uracil (HMU), but not thymine, in their genomes are susceptible to this process. In addition, we show that the photoproduct(s) in UV-irradiated DNA that produces TE by cotransfection is specific, and is not uracil, a pyrimidine dimer, thymine glycol, HMU, or a substrate for the E. coli thymine glycol DNA N-glycosylase. This photoproduct is derivable from thymine or HMU. The implications of these results are discussed.

UV-catalyzed cross-linking of Escherichia coli uracil-DNA glycosylase to DNA. Identification of amino acid residues in the single-stranded DNA binding site.

Photochemical cross-linking of Escherichia coli uracil-DNA glycosylase (Ung) to oligonucleotide dT20 was performed to identify amino acid residues that reside in or near the DNA-binding site. UV-catalyzed cross-linking reactions produced a covalent Ung x dT20 complex which was resolved from uncross-linked enzyme by SDS-polyacrylamide gel electrophoresis. Cross-link formation required native Ung and was inhibited by increasing concentrations of NaCl in a manner characteristics of NaCl inhibition of Ung catalytic activity. The Ung x dT20 complex was purified to apparent homogeneity, and mass spectrometry revealed that Ung was cross-linked to dT20 in 1:1 stoichiometry as a 31,477 dalton complex. Purified Ung x dT20 lacked detectable uracil-DNA glycosylase activity and failed to bind single-stranded DNA. Recently, we demonstrated that the bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) binds Ung and prevents further interaction with DNA (Bennett, S. E., Schimerlik, M. I., and Mosbaugh, D. W. (1993) J. Biol. Chem. 268, 26879-26885). Addition of the Ugi protein to the cross-linking reaction blocked formation of the Ung x dT20 cross-link. Conversely, the Ung x dT20 cross-link was refractory to Ugi binding. Upon trypsin digestion of Ung x dT20, four distinct products were identified as peptide x dT20 cross-links. A combination of amino acid sequence and mass spectrometric analysis revealed that four tryptic peptides (T6, T18, T19, and T18/19) were adducted to dT20. These observations suggest that dT20 is cross-linked to the Ung DNA-binding site.

Induction of O6-methylguanine-DNA-methyltransferase and N3-methyladenine-DNA-glycosylase in human cells exposed to DNA-damaging agents.

The inducibility of two DNA repair proteins, the O6-methylguanine-DNA-methyltransferase (MGMT) and the N3-methyladenine-DNA-glycosylase (ANPG), was studied by measuring the protein activities and the transcription of the MGMT and ANPG genes in a human hepatoma cell line (LICH cells). The two protein activities are enhanced after treatment with a variety of DNA-damaging agents. They are maximum 72 hr after the inducing treatments and remain elevated for about 120 hr. This induction is abolished when the cells are grown in the presence of protein or RNA synthesis inhibitors. Northern blot analysis shows that the DNA-damaging agents increase to different extents the transcription of the MGMT or ANPG genes. The transferase activity is also increased by DNA damage in a human glioblastoma cell line (T98G cells), but is not significantly modified in human normal fibroblasts, suggesting that this repair activity enhancement might occur preferentially in transformed cells, as we have previously shown for cells of rat origin. Therefore, these increased repair activities may play an important role in removing the lethal N3-methyladenine residues, the promutagenic O6-methylguanine lesions, and the potentially lethal chloroethyl adducts formed by the nitrosoureas used in cancer chemotherapy more efficiently from the cellular DNA.

[The levels of DNA repair in the cells of diploid and tetraploid cell cultures]. / Issledovanie urovnei reparatsii DNK v kletkakh diploidnykh i tetraploidnykh kletochnykh kul'tur.

Levels of unscheduled DNA synthesis after UV-irradiation were investigated in addition to the activities of DNA-repair enzyme uracil-DNA glycosilase in cells of both diploid (XC-D) and tetraploid (XC-T) cell cultures. It was shown that repair levels were increasing directly and proportionally to the cell ploidy.

[Lethal and mutagenic action of incorporated 5-3H-cytosine on extracellular phage lambda]. / Letal'noe i mutagennoe deistvie inkorporirovannogo 5-3H-tsitozina na vnekletochnyi fag lamda.

A study was made of the lethal and mutagenic effects on extracellular phage gamma of 5-3H-cytosine incorporated into DNA. The efficiencies of inactivation by incorporated 3H were equal for 5-3H-cytosine and [3H-methyl]-thymidine, but the yield of c-mutations for the former was 14 times higher. The lethal and mutagenic effects of incorporated 5-3H-cytosine did not depend on ung mutation of host cells which caused a deficiency in uracil-DNA-glycosylase. The mutagenic effect was not enhanced when SOS-repair system was induced by UV-radiation. The mutagenic effect of 5-3H-cytosine was associated with the modified mispairing bases but not with uracil residues.

Mutation by [5-3H]cytosine decay in DNA of Escherichia coli lacking uracil-DNA glycosylase activity.

The mutagenic local effect of tritium decay at the 5 position of cytosine in DNA of Escherichia coli was determined in wild-type and in ung strains defective in uracil-DNA glycosylase. In the absence of this in vivo activity any genetic consequences of uracil residues formed in DNA should be enhanced. However, the mutation frequency response was no greater in the mutant strain than in the wild type. This finding is inconsistent with the earlier suggestion that efficient production of C to T transitions by the local effect of [5-3H]cytosine decay results from the formation of uracil in cellular DNA. Some other intermediate should be considered, one that is not a substrate for uracil-DNA glycosylase.