The Drosophila mus 308 gene product, implicated in tolerance of DNA interstrand crosslinks, is a nuclear protein found in both ovaries and embryos

mus 308 designates one of over 30 mutagen sensitivity loci found in Drosophila. It is predicted to code for a 229-kDa polypeptide. Published sequence analyses of others indicate that this polypeptide would have helicase motifs near its N-terminus, and similarities to bacterial DNA polymerase I-like enzymes near its C-terminus. In our studies, two different and highly specific antibodies were prepared and used for identification as well as characterization of the mus 308 gene product. Western blot analyses reveal a single reactive polypeptide in both ovaries and embryos as well as in two Drosophila embryo tissue culture cell lines; it is nearly absent in homozygous mus 308 mutants. This polypeptide is about 229 kDa in size, and indirect immunofluorescence shows that the mus 308 gene product localizes throughout nuclei in wild-type cells but appears to be absent in a mus 308 mutant. Immunoblot analyses throughout development suggest greatest abundance at the end of embryogenesis, immediately before hatching of first instar larvae. They also showed a smaller ( approximately 100 kDa) antigenically and genetically related polypeptide found only in adult males. Immunoprecipitation, a highly effective method of specific purification, suggests that the mus 308 protein has DNA polymerase activity that is NEM-sensitive but largely aphidicolin-resistant. In addition, the immunoprecipitated material has DNA-dependent ATPase but lacks detectable helicase.     

Cloning and chromosomal mapping of the human DNA polymerase theta (POLQ), the eighth human DNA polymerase.

We have cloned the cDNA for the eighth human DNA polymerase, DNA polymerase θ. The human cDNA encodes a putative DNA polymerase of 1762 amino acids with a calculated molecular mass of 198 kDa. The derived protein sequence is homologous to the Drosophila melanogaster mus308 protein product, a putative DNA polymerase-helicase involved in repair of interstrand crosslinks. The C-terminal region contains the canonical DNA polymerase motifs A, B, and C found in the family A type of DNA polymerases, which includes Escherichia coli polymerase I. The N-terminal region contains a putative ATP binding domain but not motifs for a helicase. The gene was mapped by radiation hybrid analysis to chromosome 3q within an interval flanked by proximal marker D3S1303 and distal marker D3S3576 and, based on proximity to a gene that has been mapped cytogenetically, within band 3q13.31.     

Mus308 Mutants of Drosophila Exhibit Hypersensitivity to DNA Cross-Linking Agents and Are Defective in a Deoxyribonuclease

Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an acid-active deoxyribonuclease was observed in strains carrying the six available mutant alleles of the mus308 gene. Since that enzyme is detected at normal levels in a mutant strain that is deficient in the previously identified enzymes DNase 1 and DNase 2, it represents a new Drosophila nuclease that is designated Nuclease 3. The mus308 mutants were originally distinguished from all other mutagen-sensitive mutants of Drosophila because they exhibit hypersensitivity to the DNA cross-linking agent nitrogen mustard without expressing a concurrent sensitivity to the monofunctional agent methyl methanesulfonate. Further observations of hypersensitivity to the mutagens trimethylpsoralen, diepoxybutane and cis-platinum now establish a more general sensitivity of these mutants to agents capable of generating DNA cross-links. In spite of the hypersensitivity of the mus308 mutants to DNA cross-linking agents, the initial incision step of DNA cross-link repair is normal in mus308 cells as assayed by the alkaline elution procedure. The Drosophila mus308 mutants show promise of providing a useful model for analogous defects in other organisms including man.     

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.     

A human DNA helicase homologous to the DNA cross-link sensitivity protein Mus308.

Repair of DNA interstrand cross-links is a challenging problem for cells. Many human gene products influence sensitivity to DNA cross-linking agents, but the mechanisms of cross-link repair are unknown. In Drosophila melanogaster, the mus308 mutation leads to marked sensitivity to DNA cross-linking agents. The C-terminal portion of the Mus308 polypeptide encodes a DNA polymerase, whereas a putative DNA helicase is encoded by the N-terminal portion. As a step toward isolating proteins involved in DNA cross-link repair, we searched for mammalian genes similar to the DNA helicase portion of Mus308. Human and mouse homologs were isolated from cDNA expression libraries and designated HEL308. Human HEL308 is on chromosome 4q21 and encodes a polypeptide of 1101 amino acids. The protein was expressed in insect cells and purified. HEL308 is a single-stranded DNA-dependent ATPase and DNA helicase. Mutation of a highly conserved lysine to methionine in helicase domain I eliminated both activities. The protein readily displaces 20- to 40-mer duplex oligonucleotides. Displacement of longer substrates was less efficient but was stimulated by the single-stranded DNA-binding protein RPA. Activity was supported by ATP or dATP but not other nucleotide triphosphates. The enzyme translocates on DNA with 3' to 5' polarity and behaves as a multimer upon gel filtration.     

A large-scale screen for mutagen-sensitive loci in Drosophila

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212-mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314-mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215(ZIII-2059), that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.     

Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila

DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or “alternative” end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s) are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta), encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair.     

Characterization of the Mus308 Gene in Drosophila Melanogaster

Among the available mutagen-sensitive mutations in Drosophila, those at the mus308 locus are unique in conferring hypersensitivity to DNA cross-linking agents but not to monofunctional agents. Those mutations are also associated with an elevated frequency of chromosomal aberrations, altered DNA metabolism and the modification of a deoxyribonuclease. This spectrum of phenotypes is shared with selected mammalian mutations including Fanconi anemia in humans. In anticipation of the molecular characterization of the mus308 gene, it has been localized cytogenetically to 87C9-87D1,2 on the right arm of chromosome three. Nine new mutant alleles of the gene have been generated by X-ray mutagenesis and one was recovered following hybrid dysgenesis. Characterization of these new alleles has uncovered additional phenotypes of mutations at this locus. Homozygous mus308 flies that have survived moderate mutagen treatment exhibit an altered wing position that is correlated with reduced flight ability and an altered mitochondrial morphology. In addition, observations of elevated embryo mortality are potentially explained by an aberrant distribution of nuclear material in early embryos which is similar to that seen in the mutant giant nuclei.     

Influence of mus201 and mus308 mutations of Drosophila melanogaster on the genotoxicity of model chemicals in somatic cells in vivo measured with the comet assay

To check the possibilities of the recently developed comet assay, to be used in mechanistic studies in Drosophila melanogaster, neuroblast cells of third instar larvae are used to analyse in vivo, the effect of two repair deficient mutations: mus201, deficient on nucleotide excision repair, and mus308, deficient in a mechanism of damage bypass, on the genotoxicity of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU). The obtained results reveal: (1) MMS-induced breaks are most probably consequence of N-alkylation damage mediated abasic (AP) site breakage; (2) MMS and at least part of the EMS induced damage leading to DNA strand breaks are efficiently repaired by the nucleotide excision repair mechanism; (3) ENU and part of EMS induced damage need a functional Mus308 protein to be processed, otherwise they can lead to DNA strand breaks. In addition, the results of this work confirm the validity of neuroblast cells to conduct the comet assay, and the usefulness of this assay in in vivo mechanistic studies related to DNA repair in D. melanogaster.     

Alternate pathways of DNA replication in Escherichia coli

We have described the pcbA1 mutation which enables E. coli cells to replicate DNA in the absence of a functional dnaE gene product if DNA polymerase I (the polA gene product) is present. The pcbA1 mutation phenotypically suppresses multiple dnaEts and dnaEam alleles. The pcbA1/PolI replication pathway differs from normal in sensitivity to certain DNA-damaging agents such as methylmethane sulfonate (MMS) and a lack of damage-directed mutagenesis. We report here cloning of the pcbA1 gene in a multicopy plasmid. The pcbA1 mutation is detected only in cis; therefore, cloning necessitated gene eviction. The pcbA1 gene lies closely- linked to gyrB. We have demonstrated the physical presence of DNA polymerase I in the replicating holoenzyme complex by immunoblotting using dnaEam strains. We conclude that E. coli has two alternate replisome structures: REP-A, in which DNA polymerase I is the functional synthetic subunit; and REP-E, in which the alpha-subunit, product of the dnaE gene, is functional. To investigate further the role of individual DNA polymerases in replication, we have isolated the polB gene on multicopy plasmids.     

DNA polymerase theta purified from human cells is a high-fidelity enzyme

With the aim to identify unconventional DNA polymerases from human cells, we have set up a special assay to fractionate HeLa extracts based on the ability (i) to bypass DNA lesions, (ii) to be resistant to aphidicolin and an inhibitory antibody against pol alpha and (iii) to be non-responsive to proliferating cell nuclear antigen. After eight different chromatographic steps, an aphidicolin-resistant DNA polymerase activity was obtained that was able to utilize either undamaged or abasic sites-containing DNA with the same efficiency. Biochemical characterization and immunoblot analysis allowed its identification as the human homologue of DNA polymerase theta (hpol theta), whose cDNA has been cloned by homology with the mus308 gene of Drosophila melanogaster but still awaited detailed biochemical characterization. The purified hpol theta was devoid of detectable helicase activity, possessed a 3'-->5' exonuclease activity and showed biochemical properties clearly distinct from any other eukaryotic DNA polymerase known so far. Misincorporation and fidelity assays showed that: (i) hpol theta was able to catalyze efficiently DNA synthesis past an abasic site; and (ii) hpol theta showed high fidelity. Our findings are discussed in light of the proposed physiological role of hpol theta.     

DNA repair defects and other (mus)takes in Drosophila melanogaster.

Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA repair-related genes and to elucidate repair mechanisms. In Drosophila melanogaster, such strains are generally referred to as mutagen-sensitive (mus) mutants and currently define more than 30 genetic loci. Whereas most mus mutants have been recovered on the basis of hypersensitivity to the monofunctional alkylating agent methyl methanesulfonate, they nevertheless constitute a phenotypically diverse group, with many mutants having effects beyond mutagen sensitivity. These phenotypes include meiotic dysfunctions, somatic chromosome instabilities, chromatin abnormalities, and cell proliferation defects. Within the last few years numerous mus and other DNA repair-related genes of Drosophila have been molecularly cloned, providing new insights into the functions of these genes. This article outlines strategies for isolating mus mutations and reviews recent advances in the Drosophila DNA repair field, emphasizing mutant analysis and gene cloning.     

Characterization of a second proliferating cell nuclear antigen (PCNA2) from Drosophila melanogaster

The eukaryotic DNA polymerase processivity factor, proliferating cell nuclear antigen, is an essential component in the DNA replication and repair machinery. In Drosophila melanogaster, we cloned a second PCNA cDNA that differs from that encoded by the gene mus209 (for convenience called DmPCNA1 in this article). The second PCNA cDNA (DmPCNA2) encoded a 255 amino acid protein with 51.7% identity to DmPCNA1, and was ubiquitously expressed during Drosophila development. DmPCNA2 was localized in nuclei as a homotrimeric complex and associated with Drosophila DNA polymerase delta and epsilonin vivo. Treatment of cells with methyl methanesulfonate or hydrogen peroxide increased the amount of both DmPCNA2 and DmPCNA1 associating with chromatin, whereas exposure to UV light increased the level of association of only DmPCNA1. Our observations suggest that DmPCNA2 may function as an independent sliding clamp of DmPCNA1 when DNA repair occurs.     

A novel ochre mutation in the beta-thalassemia gene of a Thai. Identification by direct cloning of the entire beta-globin gene amplified using polymerase chain reactions

The beta-globin genes from a Thai patient compound heterozygous for beta-thalassemia and HbE disease were investigated. The 3.0-kilobase fragment containing the entire beta-globin gene was amplified by polymerase chain reaction, using Taq DNA polymerase followed by direct cloning of the amplified product into plasmid DNA. Sequence analysis of the thalassemia gene revealed only one base change, a C-A transversion within codon for an amino acid 35. This new mutation creates a premature terminator, TAA, an ochre codon, and results in a beta 0-thalassemia phenotype. The same result was obtained when this mutation was analyzed using a conventional cloning technique, direct sequencing of the amplified product, and hybridization with allele-specific oligonucleotide probes. No misincorporation was detected in the sequence analysis of the 3.0-kilobase insert of five clones of the amplified products obtained from genomic DNA of a normal individual. This approach is a rapid and accurate method for molecular cloning of the beta-globin gene and also other genes, the partial nucleotide sequences of which are known.     

Postreplication repair defects in mutants of Drosophila melanogaster

Summary Mutants of Drosophila melanogaster which are defective in DNA synthesis have been identified among mutagen-sensitive stocks through analysis of both organ and cell cultures. A new procedure employing larval brain ganglia allows poorly fertile or sterile mutants to be analyzed for the first time. Parallel studies were performed in both tissues to establish the sensitivity of the new assay relative to that of the proven cell-culture assay. Damage was induced in the DNA of cultured cells with UV irradiation and in that of ganglial cells with the carcinogen N-acetoxy-2-acetylaminofluorene. Cultures were then pulse-labeled with ³H-thymidine, incubated in the absence of thymidine, and the newly synthesized DNA was analyzed by alkaline sucrose gradient centrifugation. The molecular weight of labeled DNA from mutant cells was compared with that from control cells to assess the effect of the mutant on DNA synthesis. Among 16 mutant stocks that were scanned in either or both tissues, seven show reductions in DNA synthesis using an undamaged template. Mutants at five different genetic loci [mus(2)205, mus(3)304, mus(3)308, mus(3)310 and mus(3)311] possess a reduced capacity to synthesize DNA on a UV-damaged template in primary cell cultures. Four of these five defects can also be detected in carcinogen-treated organ cultures. Two additional defects in postreplication repair were observed with the brainganglia assay in strains that cannot be assayed in cell culture [mus(1)108, mus(2)206].Abbreviations MMS methyl methanesulfonate - HN2 nitrogen mustard - AAF 2-acetylaminofluorene - AAAF N-acetoxy-2-acetylaminofluorene - DMSO dimethyl sulfoxide     

Isolation and genetic characterisation of the Drosophila homologue of (SCE)REV3, encoding the catalytic subunit of DNA polymerase zeta

In Drosophila, about 30 mutants are known that show hypersensitivity to the methylating agent methyl methane sulfonate (MMS). Addition of this agent to the medium results in an increased larval mortality of the mutants. Using a P-insertion mutagenesis screen, three MMS-sensitive mutants on chromosome II were isolated. One of these is allelic to the known EMS-induced mus205 (mutagen sensitive) mutant. In the newly isolated mutant, a P-element is detected in region 43E by in situ hybridisation. The localisation of mus205 to this region was confirmed by deficiency mapping. The gene was cloned and shows strong homology to the Saccharomyces cerevisiae REV3 gene. The REV3 gene encodes the catalytic subunit of DNA polymerase zeta, involved in translesion synthesis. The P-element is inserted in the first exon of the mus205 gene resulting in an aberrant mRNA, encoding a putative truncated protein containing only the first 13 of the 2130 aa native Drosophila protein. The mus205 mutant is hypersensitive to alkylating agents and UV, but not to ionising radiation. In contrast to reported data, in germ cells, the mutant has no effect on mutability by X-rays, NQO and alkylating agents. In somatic cells, the mutant shows no effect on MMS-induced mutations and recombinations. This phenotype of the Drosophila mus205 mutant is strikingly different from the phenotype of the yeast rev3 mutant, which is hypomutable after UV, X-rays, NQO and alkylating agents.     

Human DNA polymerase N (POLN) is a low fidelity enzyme capable of error-free bypass of 5S-thymine glycol

Human DNA polymerase N (POLN or pol nu) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiation-induced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.     

Subunit protein-affinity isolation of Drosophila DNA polymerase catalytic subunit

gfLittle is known at present about the biochemical properties of very large-sized Drosophila DNA polymerases. In a previous study, we tried to purify Drosophila pol. catalytic subunit from embryos through seven column chromatographies and study its biochemical properties. However, we failed to characterize it precisely because an insufficient amount of the enzyme was generated. In this report, we describe direct purification from Drosophila embryos to near homogeneity using Drosophila DNA polymerase second subunit (Drosophila pol. 2) protein-conjugated affinity column chromatography and characterization of the enzyme in detail. To our knowledge this is the first demonstration of native DNA polymerase purification with activity using a subunit protein-affinity column. We observed new characteristics of Drosophila pol. catalytic subunit as follows: Drosophila pol. catalytic subunit synthesized DNA processively in the presence of both Mn(2+) and Mg(2+) ions, but Mn(2+) inhibited the 3'-5' proofreading activity, thereby decreasing the fidelity of DNA replication by 50%.