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

Abstract

A monoclonal antibody prepared against a partially purified human uracil DNA glycosylase was found, on further purification of the enzyme, to be inactive against the glycosylase. However, immunoreactivity was observed in other protein fractions that contained DNA polymerase activity. The immunoreactive protein was purified to homogeneity and identified as a catalytic subunit of DNA polymerase a by molecular mass, by aphidicolin sensitivity, and by recognition by a monoclonal antibody against human KB cell DNA polymerase a. Our monoclonal antibody had no effect on homogeneous human uracil DNA glycosylase activity but severely inhibited the activity of the homogeneous human DNA polymerase a catalytic subunit. The suspicion that the two proteins were physically associated was confirmed by finding that, on mixing the DNA polymerase a subunit with the glycosylase, the latter was strongly inhibited by our monoclonal antibody. These results demonstrate that this monoclonal antibody recognizes not only the DNA polymerase a subunit but also the uracil DNA glycosylase when it is physically attached to the polymerase subunit. These results contribute to the definition of relationships between those proteins that may comprise the human base-excision repair multienzyme complex.Recent studies have characterized the in vitro individual enzymatic reactions involved in human DNA repair. Similar studies have examined cellular DNA repair synthesis and the excision of DNA adducts in vivo (1-4). However, specific structural interrelationships between these individual proteins within multienzyme repair complexes may be an a priori requirement for the proper cellular function of individual components ofexcision repair pathways. In addition, specific alterations ofsuch physical relationships within distinct DNA repair complexes may provide a molecular mechanism for the individual cellular hypersensitivity in human genetic syndromes characterized by high rates of neoplasia (3).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). Uracil may be formed in DNA by the mutagenic deamination of cytidine (6, 7) or by the utilization of dUTP during DNA replication (8,9). The resultant apyrimidinic site would be the substrate for subsequent endonuclease incision.

Misincorporation of Uracil Into DNA as Possible Contributor to Neuronal aging and Abiotrophy

“…This finding agrees with data showing a physical association of uracil DNA-glycosylase with DNA polymerase cx (Seal & Sirover, 1986) and substantiates the hypothesis that once incorporated into DNA of adult neurons, uracil is not easily removed and might accumulate into DNA during the life span.…”

Immunological lesions in human uracil DNA glycosylase: association with Bloom syndrome.

“…As each antibody inhibited catalysis, the glycosylase-antibody complex that sediments at a higher density could not be detected (16). In contrast, this second peak of activity could be observed by using an anti-DNA polymerase a monoclonal antibody that recognizes the glycosylase when it is bound to the polymerase (16,26).…”

Determination and analysis of the complete nucleotide sequence of human herpesvirus

“…Homologs of the thymidine kinase genes found in the alpha-and gammaherpesviruses are absent in HCMV, HHV-6, and HHV-7 (17,43). The HHV-7 dUTPase and uracil-DNA glycosylase homologs presumably specify enzymatic activities involved in excision of uridine residues from DNA, by analogy to bacterial and eukaryotic counterparts (67,97,121), although there is notable sequence divergence between the HHV-7, cellular, and other herpesvirus dUTPase homologs.…”