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

A series of monoclonal antibodies has been prepared against the base excision repair enzyme uracil DNA glycosylase isolated from human placenta. Spleen cells from BALB/c mice immunized with purified human placental uracil 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.     

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

Three monoclonal antibodies that react with uracil DNA glycosylase of normal human placenta were tested to determine whether one of the antibodies could be used as a negative marker for Bloom syndrome. As defined by enzyme-linked immunosorbent assay, monoclonal antibody 40.10.09, which reacts with normal human glycosylase, neither recognized nor inhibited native uracil DNA glycosylase from any of five separate Bloom syndrome cell strains. Immunoblot analyses demonstrated that the denatured glycosylase protein from all five Bloom syndrome cell strains was immunoreactive with the 40.10.09 antibody. Further, each native enzyme was immunoreactive with two other anti-human placental uracil DNA glycosylase monoclonal antibodies. In contrast, ELISA reactivity was observed with all three monoclonal antibodies in reactions of glycosylases from 5 normal human cell types and 13 abnormal human cell strains. These results experimentally verify the specificity of the aberrant reactivity of the Bloom syndrome uracil DNA glycosylase. The possibility arises that determination of the lack of immunoreactivity with antibody 40.10.09 may have value in the early diagnosis of Bloom syndrome.     

A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase.

We have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein (Mr 66,000) that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a beta-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated Mr 36,050 and pI 8.7, corresponding to the Mr 37,000 and pI 8.1 of purified human placental UDG. No homology was seen between this cDNA and the UDG of herpes simplex virus, Escherichia coli, and yeast; nor was there homology with the putative human mitochondrial UDG cDNA or with a second human nuclear UDG cDNA. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated from it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.     

Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA.

T4 endonuclease V [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1)], which is involved in repair of UV-damaged DNA, has been purified to apparent physical homogeneity. Incubation of UV-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. The activity to produce alkali-labile sites was optimal in a relatively broad pH range (pH 6.0-8.5), whereas the activity to form nicks had a narrow optimum near pH 6.5. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity [endodeoxyribonuclease (apurinic or apyrimidinic, EC 2.1.25.2]. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. An enzyme fraction from cells infected with bacteriophage T4v1, a mutant that is sensitive to UV radiation, was defective in both glycosylase and endonuclease activities. Moreover, occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities, and suppression of the mutation rendered both activities partially active. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4. Because the two activities exhibited different thermosensitivity, it was further suggested that conformation of the active sites for these activities may be different.     

Primary structure of the catalytic subunit of human DNA polymerase delta and chromosomal location of the gene.

The catalytic subunit (Mr approximately 124,000) of human DNA polymerase delta has been cloned by PCR using poly(A)+ RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA polymerase delta. The human cDNA was 3443 nucleotides in length and coded for a polypeptide of 1107 amino acids. The enzyme was 94% identical to bovine DNA polymerase delta and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes. The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end. The gene coding for human DNA polymerase delta was localized to chromosome 19.     

The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase

The major DNA photoproduct of dormant, UV-irradiated Bacillus subtilis spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)]. During spore germination, SP is reversed to two intact thymines in situ by the DNA repair enzyme SP lyase, an S-adenosylmethionine (S-AdoMet)-dependent iron-sulfur ([Fe-S]) protein encoded by the splB gene. In the present work, cross-linking, SDS/PAGE, and size exclusion chromatography revealed that SplB protein dimerized when incubated with iron and sulfide under anaerobic reducing conditions. SplB isolated under aerobic conditions generated an EPR spectrum consistent with that of a partially degraded [3Fe-4S] center, and reduction of SplB with dithionite shifted the spectrum to that of a [4Fe-4S] center. Addition of S-AdoMet to SplB converted some of the [4Fe-4S] centers to an EPR-silent form consistent with electron donation to S-AdoMet. HPLC and electrospray ionization MS analyses showed that SP lyase cleaved S-AdoMet to generate 5'-deoxyadenosine. The results indicate that (i) SP lyase is a homodimer of SplB; (ii) dimer formation is coordinated by a [4Fe-4S] center; and (iii) the reduced [4Fe-4S] center is capable of donating electrons to S-AdoMet to generate a 5'-adenosyl radical that is then used for the in situ reversal of SP. Thus, SP lyase belongs to the "radical SAM" superfamily of enzymes that use [Fe-S] centers and S-AdoMet to generate adenosyl radicals to effect catalysis. SP lyase is unique in being the first and only DNA repair enzyme known to function via this novel enzymatic mechanism.     

Identification of a poxvirus gene encoding a uracil DNA glycosylase.

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.     

Proofreading by the epsilon subunit of Escherichia coli DNA polymerase III increases the fidelity of calf thymus DNA polymerase alpha.

Addition of the 3'----5' proofreading exonuclease, epsilon subunit of Escherichia coli DNA polymerase III, to DNA polymerase alpha from calf thymus has been studied. Alone, calf thymus DNA polymerase alpha terminates in vitro DNA synthesis upon insertion of noncomplementary nucleotides. Upon addition of the epsilon subunit, DNA polymerase alpha elongates the newly synthesized DNA as a result of hydrolysis of the 3'-terminal mispair. The fidelity of DNA polymerase alpha in vitro is increased 7-fold by addition of the exonuclease. The functional interaction between DNA polymerase alpha and the epsilon subunit is independent of any detectable physical association. This suggests that a mechanism for proofreading could exist in mammalian cells involving sequential catalysis by DNA polymerase alpha excision of errors by a separate 3'----5' exonuclease, and further elongation onto correctly base-paired 3' termini by DNA polymerase alpha.     

Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment.

A human 70-kDa heat shock protein (hsp70) gene segment has been isolated. The segment contains 3.15 kilobase pairs (kbp) of 5' nontranscribed sequence, an RNA leader of 119 bp, and a protein-coding region of 741 bp. The human protein sequence shows a high degree of homology to hsp70 sequences from other species. Expression experiments in Xenopus oocytes and mammalian cells indicate that a region that includes only 105 bp of 5' nontranscribed sequence contains all elements required for the efficient heat-controlled expression of the human gene. Two adjacent identical sequence elements, which are partly homologous to the Drosophila "heat shock consensus" sequence, are located 57 to 76 bp upstream from the capping site. Interestingly, the capping site itself is flanked by inverted repeat sequences.     

Organization of the multiple genes for the 70,000-dalton heat-shock protein in Drosophila melanogaster.

The organization and number of 70,000-dalton heat-shock protein genes of Drosophila melanogaster has been investigated in a wild-type Oregon R fly stock and in a KC cell line. Six copies were found in the KC cells, and slightly more were found in the Oregon R population examined. In both cases, the basic gene element consisting of the mRNA coding region plus a short 5' "noncoding" sequence element was conserved. Two gene variants distinguished by specific restriction sites were found in both genomic DNAs. Restriction maps of the six genes in KC cells showed that these two gene variants are arranged differently. Restriction analysis of Oregon R embryonic DNA revealed polymorphism in the organization of the genes, which is not observed in KC cells. The data suggest that the arrangement as well as the number of genes for the 70,000-dalton heat-shock protein in D. melanogaster is subject to variations at both the 87A and 87C cytogenetic loci.     

Unraveling the three-metal-ion catalytic mechanism of the DNA repair enzyme endonuclease IV

Endonuclease IV belongs to a class of important apurinic/apyrimidinic endonucleases involved in DNA repair. Although a structure-based mechanistic hypothesis has been put forth for this enzyme, the detailed catalytic mechanism has remained unknown. Using thermodynamic integration in the context of ab initio quantum mechanics/molecular mechanics molecular dynamics, we examined certain aspects of the phosphodiester cleavage step in the mechanism. We found the reaction proceeded through a synchronous bimolecular (A(N)D(N)) mechanism with reaction free energy and barrier of -3.5 and 20.6 kcal/mol, in agreement with experimental estimates. In the course of the reaction the trinuclear active site of endonuclease IV underwent dramatic local conformational changes: shifts in the mode of coordination of both substrate and first-shell ligands. This qualitative finding supports the notion that structural rearrangements in the active sites of multinuclear enzymes are integral to biological function.     

Assignment of the gene for human DNA polymerase alpha to the X chromosome.

We have applied an assay based on a monoclonal antibody that discriminates the activity of human DNA polymerase alpha in rodent-human somatic cell hybrid clones to identify a single genetic locus that is both necessary and sufficient for the expression of DNA polymerase alpha. We have mapped this locus to the short arm of the human X chromosome, near the junction of bands Xp21.3 and Xp22.1, and demonstrated that it is not expressed from an inactive X chromosome.     

Intracellular localization of DNA polymerase alpha.

Immunoglobulin (IgG) and the F(ab')2 fragment of IgG were prepared from serum of a rabbit immunized with purified calf thymus DNA polymerase alpha (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7). An indirect immunofluorescent method based on these reagents was used to detect the intracellular localization of DNA polymerase alpha in primary fetal bovine fibroblasts. The results show that the bulk of DNA polymerase alpha is located in the perinuclear region of the cytoplasm. Immunofluorescent staining of cytoplast and Ficoll-Paque gradient-purified karyoplast fragments resulting from cytochalasin enucleation show the presence of DNA polymerase alpha in cytoplasts and the virtual absence of the enzyme in the nucleus of the karyoplast itself. The implication of this unusual intracellular location for DNA polymerase alpha is discussed.     

Mismatch-specific thymine DNA glycosylase and DNA polymerase beta mediate the correction of G.T mispairs in nuclear extracts from human cells.

To avoid the mutagenic effect of spontaneous hydrolytic deamination of 5-methylcytosine, G.T mispairs, arising in DNA as a result of this process, should always be corrected to G.C pairs. We describe here the identification of a DNA glycosylase activity present in nuclear extracts from HeLa cells, which removes the mispaired thymine to generate an apyrimidinic (AP) site opposite the guanine. We further show, using a specific antibody and inhibitors, that the single nucleotide gap, created upon processing of the AP site, is filled in by DNA polymerase beta. This finding substantiates the proposed role of this enzyme in short-patch DNA repair.     

Cloning a eukaryotic DNA glycosylase repair gene by the suppression of a DNA repair defect in Escherichia coli.

If eukaryotic genes could protect bacteria with defects in DNA repair, this effect could be exploited for the isolation of eukaryotic DNA repair genes. We have thus cloned a DNA repair gene from Saccharomyces cerevisiae that directs the synthesis of a DNA glycosylase that specifically releases 3-methyladenine from alkylated DNA and in so doing protects alkylation-sensitive Escherichia coli from killing by methylating agents. The cloned yeast gene was then used to generate a mutant strain of S. cerevisiae that carries a defect in the glycosylase gene and is extremely sensitive to DNA methylation. This approach may allow the isolation of a large number of eukaryotic DNA repair genes.     

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.6.1.8) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.     

Specific association between the human DNA repair proteins XPA and ERCC1.

Processing of DNA damage by the nucleotide-excision repair pathway in eukaryotic cells is most likely accomplished by multiprotein complexes. However, the nature of these complexes and the details of the molecular interactions between DNA repair factors are for the most part unknown. Here, we demonstrate both in vivo, using the two-hybrid system, and in vitro, using recombinant proteins, that the human repair factors XPA and ERCC1 specifically interact. In addition, we report an initial determination of the domains in ERCC1 and XPA that mediate this interaction. These results suggest that XPA may play a role in the localization or loading of an incision complex, composed of ERCC1 and possibly other repair factors, onto a damaged site.