Steven A. Leadon

Monoclonal antibody to DNA containing thymine glycol

Exposure of DNA to ionizing or near ultraviolet radiation modifies thymine to form ring-saturated products. One of the major products formed is 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol). Thymine glycol can also be selectively formed by oxidizing DNA with OsO4. We have isolated hybrids that produce monoclonal antibodies against thymine glycol by fusing mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells from BALB/c mice immunized with OsO4-oxidized poly(dT) complexed with methylated bovine serum albumin. This report describes the characterization of the antibody from one hybridoma using a competitive enzyme-linked immunosorbent assay (ELISA). The antibody reacted with both single- and double-stranded DNA treated with OsO4, and with OsO4-treated poly(dA-dT) and poly(dT); it did not crossreact with unmodified or apurinic DNA. It also reacted with DNA treated with H2O2 or with gamma-rays at doses as low as 250 rad. We were able to detect 2 fmoles of thymine glycol in OsO4-treated DNA and could quantitate 1 thymine glycol per 220 000 thymines. Using the antibody and the ELISA, the formation and removal of thymine glycol was examined in cultures of African green monkey cells irradiated with 25 krad of gamma-rays. The antibody reactive sites produced by irradiation (8.5 per 10(6) thymines) were efficiently removed from the cellular DNA.

Enhanced transforming activity of pSV2 plasmids in human cells depends upon the type of damage introduced into the plasmid

When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.

Cell-cycle-dependent repair of damage in alpha and bulk DNA on monkey cells

Excision repair of bulky chemical adducts in alpha DNA of confluent cultures of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have compared the removal of adducts produced by treatment with aflatoxin B1 (AFB1) and N-acetoxy-2-acetylamino-fluorene (NA-AAF) from alpha DNA sequences in synchronized and exponentially growing cultures of monkey cells. Proficient removal of AFB1 adducts in alpha DNA was observed in exponentially growing cultures. However, as the cultures approached confluence, adduct removal from alpha DNA became deficient. Cells synchronized by subculturing confluent cultures exhibited proficient removal of adducts from both alpha and bulk DNA when treated in early G1 or late S/G2 while those cells treated in early S phase did not remove adducts from either alpha or bulk DNA. We conclude that the accessibility of chemical adducts to repair in alpha chromatin is influenced by the growth state and the cell cycle stage.

Enhanced transforming activity of ultraviolet-irradiated pSV2-gpt is due to damage outside the gpt transcription unit

We have shown that when pSV2-gpt is introduced into human cells by calcium phosphate coprecipitation, the yield of Gpt+ transformants is increased by irradiating the plasmid with 254 nm uv. To elucidate the mechanism underlying this response, we constructed pSV2-gpt molecules in which the uv damage was confined to a particular region: a 3.0-kb region containing the pBR322 sequences and simian virus 40 (SV40) sequences not required for expression of the gpt gene, or a 2.3-kb fragment containing the Escherichia coli gpt gene together with the SV40 early promoter and sequences needed for splicing and polyadenylation. The transforming activity of the plasmid was greatly enhanced by uv damage confined to the 3.0-kb pBR322 region and increased linearly with uv dose up to 1 kJ/m2, but remained relatively constant at doses between 2 and 8 kJ/m2. Positioning the damaged region upstream, or both upstream and downstream, from the gpt transcription unit increased the uv enhancement slightly compared to positioning the damaged region only downstream. In contrast, transforming activity was significantly decreased by damage in the 2.3-kb gpt transcription unit. These results suggest that uv damage outside a selectable marker gene in a plasmid can increase the probability of stable integration of the plasmid into the genome of recipient cells without inhibiting expression of of the gene.