Paul V. Harris

Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.

Mutants partially defective in excision repair at five autosomal loci in Drosophila melanogaster

Primary cell cultures derived from mutants in seventeen different genes were analyzed for their ability to excise pyrimidine dimers from DNA. Five of these mutagen-sensitive mutants [mus(2)205A1, mus(3)302D1, mus(3) 304D3, mus(3)306D1, mus(3)308D2] display a significantly reduced excision capacity relative to control cultures. In addition, two of the five [mus(3)306D1, mus(3)308D2] are defective in the accumulation of single-strand breaks normally seen after ultraviolet irradiation. This study, therefore, brings the total number of Drosophila mutants known to be defective in excision repair to seven. The results are discussed relative to other genetic and biochemical properties of these mutants.

A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764-5771.] reported the molecular cloning of Drosophila mus308 gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308 flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophila replication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template-primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophila polymerase classes such as alpha, beta, gamma, delta, epsilon and zeta and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3-5 exonuclease activities as shown by the chromatographies.

Excision repair in Drosophila. Analysis of strand breaks appearing in DNA of mei-9 mutants following mutagen treatment.

Excision repair of DNA damage has been analyzed in primary and established cell cultures of Drosophila melanogaster. Chemical and enzymatic assays for pyrimidine dimers reveal a strong deficiency in dimer excision from cells which are mutant at the mei-9 locus. Single-strand interruptions, which appear in high molecular weight DNA after ultraviolet irradiation of control cells have been monitored by alkaline elution. The appearance of such breaks is greatly enhanced by inhibitors of DNA synthesis. In mutant mei-9D2 cells, on the other hand, the level of ultraviolet-induced breaks is much reduced and inhibitors fail to potentiate the response. These results imply that the inhibitors cause an accumulation of the transient strand interruptions that normally occur in excision repair by reducing the rate of the resynthesis step. Failure of the mei-9D2 cells to accumulate such intermediates strongly suggests that the initial nicking never occurs in these cells. Confirmatory experiments have also been performed with the alternate mutagen N-acetoxy-N-acetyl-2-aminofluorene.

A MITOCHONDRIAL NUCLEASE IS MODIFIED IN DROSOPHILA MUTANTS (MUS308) THAT ARE HYPERSENSITIVE TO DNA CROSSLINKING AGENTS

SummaryThe mus308 mutants of Drosophila have previously been demonstrated to be defective in an enzyme that is designated Nuclease 3 (Boyd et al. 1990b). In this study that enzyme is shown to be present in mitochondria of both wild-type flies and embryos. Since the mus308 mutants are hypersensitive to DNA crosslinking agents, Nuclease 3 is potentially required for resistance of the mitochondrial genome to such agents. In support of this hypothesis, electron microscopic studies of mus308 mutant flies that had been exposed to nitrogen mustard revealed an increased frequency of mitochondrial abnormalities. Further investigation of the defect at the enzymological level revealed that the mutants possess a new nuclease activity that is apparently a modified form of the wild-type protein. In the earlier study, enzyme extracts from mus308 mutants were found to lack an enzyme with a pl of approximately 6.2. More precisely defined assay conditions in this study revealed the appearance of a new nuclease activity with a higher pI in extracts from mutants. This observation, together with the finding that only the normal enzyme form is present in heterozygous individuals, supports the hypothesis that the mus308 locus is not the structural gene for the enzyme. Rather, the mus308 gene product is necessary for Nuclease 3 to assume the lower pI. Nuclease 3 has been partially purified and characterized from wild-type embryos. Its activity is stimulated by Mg++ and ATP. Optimum activity is found at a pH of 5.5 and a NaCl concentration of 50–100 mM. Nuclease 3 exhibits a temperature optimum of 42°C and is insensitive to N-ethylmaleimide. The enzyme is probably membrane-associated because it exhibits a strong tendency to aggregate and detergent is required for full solubilization.

Repair of double-strand DNA breaks in Drosophila

This study describes the repair kinetics of DNA double-strand breaks in primary and established cell cultures of Drosophila melanogaster. Double-strand breaks, induced by X-irradiation, were monitored by neutral elution. In primary cell cultures 50% of the double-strand breaks induced by 10 kR of X-rays are repaired within 45 min and 80% of the breaks are repaired within 2-3 h. Repair kinetics in established cell cultures are similar; 50% of the induced breaks are repaired within 20 min and 88% within 3 h. Mutants deficient in other types of DNA repair were also assayed for their capacity to repair double-strand breaks.

A possible functional role for a new class of eukaryotic DNA polymerases

The recent Correspondence from Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1] regarding a new family of eukaryotic proteins similar to bacterial DNA polymerase I raises a number of interesting questions. Our report on the characterization of the Drosophila DNA repair gene mus308[2xMolecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Harris, PV, Mazina, OM, Leonhardt, EA, Case, RB, Boyd, JB, and Burtis, KC. Mol Cell Biol. 1996; 16: 5764–5771PubMedSee all References[2] and a related Caenorhabditis elegans gene addresses several of these questions, including the possible role of this gene family.The mus308 gene (Genbank L76559) encodes a 229 kDa polypeptide, the carboxy-terminal domain of which is closely related to the polymerase domain of the bacterial DNA polymerase I enzymes. Remarkably, the amino-terminal domain of the Mus308 polypeptide also includes the seven amino-acid sequence motifs that are characteristic of the ‘superfamily 2’ DNA and RNA helicases [3xTwo related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Gorbalenya, AE, Koonin, EV, Donchenko, AP, and Blinov, VM. Nucleic Acids Res. 1989; 17: 4713–4730Crossref | PubMed | Scopus (679)See all References[3]. This finding makes mus308 not only the first characterized gene encoding a eukaryotic homolog of the bacterial DNA polymerase I family, but also the first reported gene encoding both helicase and polymerase motifs in a single polypeptide.A gene very similar to mus308, which we have designated mus-1, encoded by genomic sequences in the cosmids R12B2 (U00066) and W03A3 (U50184), is predicted to exist in C. elegans. A combination of genomic and partial cDNA sequences indicate that the carboxy-terminal domain of the Mus-1 polypeptide is highly homologous to the polymerase domains of Mus308 and other members of the DNA polymerase I family [2xMolecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Harris, PV, Mazina, OM, Leonhardt, EA, Case, RB, Boyd, JB, and Burtis, KC. Mol Cell Biol. 1996; 16: 5764–5771PubMedSee all References[2], as noted by Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1]. Upon comparing the remainder of the mus308 coding sequences and C. elegans genomic sequences located upstream of the Mus-1 polymerase domain, a helicase domain closely related to the Mus308 helicase domain was immediately apparent [2xMolecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Harris, PV, Mazina, OM, Leonhardt, EA, Case, RB, Boyd, JB, and Burtis, KC. Mol Cell Biol. 1996; 16: 5764–5771PubMedSee all References[2]. Further limited sequence homology between the Mus308 and predicted Mus-1 polypeptides was also seen in the region between the helicase and polymerase domains (P.H., unpublished observations). The juxtaposition of Mus308-related helicase and polymerase domains and sequence similarities in the region between the two domains strongly support the hypothesis that the mus-1 gene encodes a protein homologous to Mus308. The existence of an evolutionarily conserved enzyme family with this unique combination of helicase and polymerase motifs suggests a conserved functional role in DNA repair for these proteins. Direct evidence for such a role is available, however, only for Mus308 [4xThird-chromosome mutagen-sensitive mutants of Drosophila melanogaster. Boyd, JB, Golino, MD, Shaw, KE, Osgood, CJ, and Green, MM. Genetics. 1981; 97: 607–623PubMedSee all References[4], which is required to repair the damage caused by mutagens such as nitrogen mustard and cisplatin that result in interstrand cross-links [5xmus308 mutants of Drosophila exhibit hypersensitivity to DNA cross-linking agents and are defective in a deoxyribonuclease. Boyd, JB, Sakaguchi, K, and Harris, PV. Genetics. 1990; 125: 813–819PubMedSee all References[5].As reported by Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1], two distinct human sequences encoding polypeptides with similarity to the polymerase domain of the bacterial DNA polymerase I family are also present in the sequence databases. We have also noted the existence of these coding sequences, which encode polypeptides related to the Mus308 polymerase domain. The sequence most closely related to mus308 is represented by human cDNA clone za38h12.r1 (Genbank W00829. Sequence information from partial cDNAs and RT-PCR products derived from this gene show additional homology to mus308 in sequences upstream of the polymerase domain (P.V.H. and K.C.B., unpublished observations), suggesting homology to the mus308/mus-1 family; however, the presence of helicase motifs remains to be established. We have also noted the sequences homologous to the Mus308 polymerase domain in the Huntingtons disease region of the genome, as reported by Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1]. In this case, however, the availability of almost 2 megabases of sequence information upstream of this domain reveals the absence of any recognizable helicase motifs (P.V.H. and K.C.B., unpublished observations), suggesting that this gene is not likely to be a functional homolog of the mus308/mus-1 family.Sonnhammer and Wootton [1xWidespread eukaryotic sequences, highly similar to bacterial DNA polymerase I, looking for functions. Sonnhammer, EL and Wootton, JC. Curr Biol. 1997; 7: R463–R465Abstract | Full Text | Full Text PDF | PubMedSee all References[1] note that it is ‘tempting to consider DNA repair’ as a possible function of these DNA polymerases, but that ‘experimental verification of polymerase activity is the next step.’ Our previous report [2xMolecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Harris, PV, Mazina, OM, Leonhardt, EA, Case, RB, Boyd, JB, and Burtis, KC. Mol Cell Biol. 1996; 16: 5764–5771PubMedSee all References[2] indeed confirms a role in DNA repair for at least the prototypical member of this new gene family, mus308. Furthermore, we have recently determined that the polymerase domain of the polypeptide encoded by the za38h12.r1 human cDNA possesses enzymatic polymerase activity, confirming the prediction based on amino-acid sequence (P.V.H. and K.C.B., unpublished observations). In the light of this enzymatic activity, we have termed this new human DNA polymerase gene POLH. The next step is to determine the functional role of the polymerase (and possibly helicase) domains of this new class of eukaryotic DNA polymerases in DNA repair and other cellular functions.

Alteration of a nuclease in Fanconi anemia

Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a pI of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.

X-Ray sensitivity and single-strand DNA break repair in mutagen-sensitive mutants of Drosophila melanogaster

Mutants of Drosophila melanogaster that are sensitive to chemical mutagens were analyzed for sensitivity to X-rays and for the capacity to repair single-strand DNA breaks induced by X-rays. Analysis of X-ray sensitivity demonstrated that 74% of the mutants assayed display some X-ray sensitivity, with 75% of the sensitive lines being extremely sensitive. Repair of single-strand breaks was assayed after both high and low doses of irradiation in order to permit detection of repair over a wide range of damage. The results of this investigation fail to show a correlation between X-ray sensitivity and this particular repair process. Repair of single-strand breaks is therefore mediated by repair processes unrelated to those that are disrupted in the current mutant collection.

Effects of radiation on the survival of excision-defective cells fromDrosophila melanogaster

The effect of various doses of ultraviolet light and ionizing radiation on the survival of excision-defective Drosophila cells has been determined by cloning treated and untreated cells in agarose. Although excision-defective cells survive moderate amounts of DNA damage, they display a severe hypersensitivity to both types of radiation relative to excision-proficient cells. Exposure of ultraviolet-irradiated cells to fluorescent light results in a reduction of the density of pyrimidine dimers in cellular DNA and a 10- to 20- fold increase in survival. Parallel analysis of dimer density and survival, however, suggests that much of the lethal effect of ultraviolet light is due to nondimer damage. Cell proliferation was monitored in both excision-proficient and excision-defective cells exposed to doses of ultraviolet light that reduced survival by 90%. Under these conditions excision-proficient cells displayed exponential growth whereas excision-defective cells exhibited no cell proliferation for 12 days.