A novel protein complex distinct from mismatch repair binds thioguanylatedDNA

To elucidate molecular mechanism(s) of cellular response to mercaptopurine, a widely used antileukemic agent, we assessed mercaptopurine (MP) sensitiv ity in mismatch repair (MMR) proficient and MMR deficient human acute lymph oblastic leukemia (ALL) cells. Sensitivity to thiopurine cytotoxicity was n ot dependent on MMR (i.e., MutS alpha) competence among six cell lines test ed. Using electrophoretic mobility shift assay analysis, we found that the incubation of nuclear extracts from ALL cells with synthetic 34-mer DNA dup lexes containing deoxythioguanosine (G(S)) within either G(S).T or G(S).C p airs, resulted in formation of a DNA-protein complex distinct from the DNA- MutS alpha complex and unaffected by ATP. Isolation and sequence analysis o f proteins involved in this DNA-protein complex identified glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) as a component. Western blot analysis of n uclear extracts from a panel of human lymphoblastic leukemia cell lines rev ealed markedly different basal levels of GAPDH in nuclei, which was signifi cantly related to thiopurine sensitivity (p = 0.001). Confocal analysis rev ealed markedly different intracellular distribution of GAPDH between nucleu s and cytosol in six human ALL cell lines. Redistribution of GAPDH from cyt osol to nucleus was evident after MP treatment. These findings indicate tha t a new DNA-protein complex containing GAPDH and distinct from known MMR pr otein-DNA complexes binds directly to thioguanylated DNA, suggesting that t his may act as a sensor of structural alterations in DNA and serve as an in terface between these DNA modifications and apoptosis.