Journal
DNA REPAIR
Volume 4, Issue 9, Pages 971-982Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.dnarep.2005.04.020
Keywords
DNA repair; interstrand crosslink repair; human DNA polymerase theta; nucleus; mus308; Drosophila
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Funding
- NIEHS NIH HHS [ES04068] Funding Source: Medline
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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.
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