Human DNA damage checkpoint protein hRAD9 is a 3 ' to 5 ' exonuclease

Human RAD9 protein (hRAD9) is a homolog of the fission yeast Rad9 protein, one of the six so-called checkpoint Rad proteins involved in the early step s of DNA damage checkpoint response in Schizosaccharomyces pombe. It has be en shown previously that, in vivo, a highly modified form of hRAD9 makes a ternary complex with two other checkpoint Rad proteins, hRAD1 and hHUS1 (Vo lkmer, E., and Karnitz, L, M. (1999) J. Biol. Chem. 274, 567-570; St. Onge, R. P., Udell, C. M., Casselman, R., and Davey, S. (1999) Mel. Biol Cell. 1 0, 1985-1995), However, the function of this complex is not known at presen t. To help define the functions of checkpoint Rad proteins in humans, we ex pressed hRAD9 in Escherichia coil, purified the recombinant protein and cha racterized it. We found that hRAD9 is a 3' to 5' exonuclease and located th e nuclease active site to the region between residues 51 and 91 of the 391- amino acid-long protein. Our results suggest that exonucleolytic processing of primary DNA lesion by hRAD9 may contribute to DNA damage checkpoint res ponse in humans.