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.
Pang
Maria
M
Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA.
McConnell
Maeve
M
Fisher
Paul A
PA
D000818:Animals; D002458:Cell Fractionation; D003432:Cross-Linking Reagents; D004256:DNA Polymerase I / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D045643:DNA Repair Enzymes; D004259:DNA-Directed DNA Polymerase; D029721:Drosophila Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000737:chemistry; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015151:Immunoblotting; D047468:Immunoprecipitation; D008297:Male; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D010053:Ovary / Q000737:chemistry*; D014461:Ultracentrifugation; D000097462:DNA Polymerase theta
D003432:Cross-Linking Reagents; D004256:DNA Polymerase I; D045643:DNA Repair Enzymes; D004259:DNA-Directed DNA Polymerase; D029721:Drosophila Proteins; D009687:Nuclear Proteins; C101782:DNApol-theta protein, Drosophila; D000097462:DNA Polymerase theta