Isolation and characterization of monoclonal antibodies directed against the DNA repair enzyme uracil DNA glycosylase from human placenta.

Abstract

A series of monoclonal antibodies has been prepared against the base excision repair enzyme uracit DNA glycosylase isolated from human placenta. Spleen cells from BALB/ c mice immunized with purified human placentaluracil DNA glycosylase were fused with either P3X63 Ag8.653 or SP2/0 myeloma cells. Hybridomas producing antibodies directed against the placental glycosylase were identified in an enzyme-linked immunosorbent assay. Each positive hybridoma was cloned twice by limit dilution and tested for anti-glycosylase activity in an enzyme immunoprecipitation assay. Each of the four clones examined in detail precipitated enzyme activity in an immunoprecipitation reaction only in the presence of rabbit anti-mouse IgG as a second antibody. No anti-uracil DNA glycosylase activity was observed in a spontaneous hybridoma used as a control. Each monoclonal antibody immunoprecipitated uracil DNA glycosylases isolated from several human tissues. Partial crossreactivity was observed with rat liver glycosylase and with a hamster enzyme. In contrast, no crossreactivity was observed with yeast or Escherichia coli glycosylase. Glycerol gradient sedimentation analysis demonstrated that one of the antibodies bound to the glycosylase at a site that did not diminish its catalytic activity. A second monoclonal antibody bound at a determinant that affected catalytic activity. Analysis of antibody-glycosylase interactions suggests that human cells contain antigenically distinct glycosylase species that may be encoded by individual uracil DNA glycosylase genes. The potential use of these monoclonal antibodies in studies examining the regulation of.glycosylase isoenzymes during cell proliferation in normal human cells and in cells from cancer-prone individuals is considered.Recent studies have indicated that there is a correlation between the proliferative state of a eukarvotic cell and the capacity of that cell to repair DNA after chemical or physical insult. Increases in the.repair capacity of proliferating cells above basal levels present in quiescent cells have been observed for all DNA repair pathways that have been examined. These have included nueleotide excision repair after UV irradiation (1-6) or after exposure to N-acetoxyacetylaminofluorene (2, 6), base excision repair after exposure to methyl methanesulfonate (5, 6) or to sodium bisulfite (5), and repair after exposure to ionizing radiation (7). This enhancement of repair capacity has been observed with both asynchronous and synchronous cell populations, using various protocols to quantitate DNA repair. The in vitro quantitation of individual DNA repair enzyme activities during cell proliferation demonstrated that increases in the spe-

Physical association of the human base-excision repair enzyme uracil DNA glycosylase with the 70,000-dalton catalytic subunit of DNA polymerase alpha.

“…Several eukaryotic DNA polymer- 16.11.08 (95 Ag) for 120 min at 40C. The enzyme/antibody mixture was then sedimented through a 5-ml 10-35% glycerol gradient as described (5). Highly purified Bacillus subtilis uracil DNA glycosylase was a generous gift of Nahum Duker.…”

“…Human placental uracil DNA glycosylase was partially purified through DEAE-cellulose, phosphocellulose, and hydroxylapatite column chromatography as described (5 centrifugation at 2300 x g for 10 min at 4°C. The precipitate was redissolved and reprecipitated twice using 1 ml of 0.2 mM NaOH and 1 ml of 10% trichloroacetic acid, sequentially, followed by centrifugation as described above.…”

“…The position of the polymerase subunit was also altered to sediment similarly to the glycosylase. The partial association of this glycosylase with the polymerase subunit conforms with the partial cross-reactivity of that glycosylase, as determined by enzyme immunoprecipitation (5).…”

“…To examine the structural associations required for the activity of multienzyme DNA repair pathways, we prepared a series of monoclonaf antibodies using partially purified human placental uracil DNA glycosylase as the antigen (5). The uracil DNA glycosylase removes uracil residues from DNA as an initial step of base-excision repair (1,2).…”

Isolation and characterization of the human uracil DNA glycosylase gene.

“…A series of monoclonal antibodies was prepared against the partially purified human placental uracil DNA glycosylase (17). Subsequent analysis showed that each antibody recognized determinants on the homogeneous placental enzyme (18).…”