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(PP325) Role of Rad9 in base excision repair.
Balajee, Adayabalam1, Hopkins, Kevin1, Lieberman, Howard*,1, 1 Center for Radiological Research, New York, NY, USA
ABSTRACT- The Rad9 gene is evolutionarily conserved, and homologues have been found in fission yeasts (rad9), mice (Mrad9) and humans (HRAD9), as well as other organisms. The encoded proteins promote resistance to DNA damage and regulate cell cycle checkpoints, but details of their functions are not fully defined. We constructed Mrad9 mutant mouse ES cells to determine the function of the gene in mammals. We found that, relative to Mrad9+/+ cells, the Mrad9-/- population is highly sensitive to ionizing radiation, UV light, hydroxyurea, cisplatin, and several alkylating agents, including methylmethane sulfonate as well as ethylmethane sulfonate, thus indicating that the gene promotes cellular resistance to these agents. To define the molecular basis for these sensitivities, we initiated studies to characterize DNA repair in these cells. We used cell-free extracts from Mrad9+/+ and Mrad9-/- cells, in combination with oligos containing site specific damage, to test whether the protein is important for incision in DNA at minor adducts that usually serve as substrates for base excision repair (BER). Thus far, we found that incision at tetrahydrofuran-abasic sites and etheno-adenosine occurred efficiently in both extracts. However, incision at sites of DNA containing uracil was clearly defective in the Mrad9-/- extracts. For example, after 2 hrs at 37°C, wild-type extracts incised 42% of the damaged oligos, whereas the mutant only processed 15%. These results indicate that Rad9 participates in BER for mending some but not all types of DNA damage potentially capable of being repaired by this mechanism. Studies are underway to clarify the role of Rad9 in DNA repair. °
Key words: base excision repair, Rad9, DNA repair, uracil
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