The Drosophila mus308 gene product, implicated in tolerance of DNA interstrand crosslinks, is a nuclear protein found in, both ovaries and embryos

mus308 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 mus308 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 mus308 mutants. This polypeptide is about 229 kDa in size, and indirect immunofluorescence shows that the mus308 gene product localizes throughout nuclei in wild-type cells but appears to be absent in a mus308 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 (similar to 100kDa) antigenically and genetically related polypeptide found only in adult males. Immunoprecipitation, a highly effective method of specific purification, suggests that the mus308 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. (c) 2005 Elsevier B.V. All rights reserved.