Analysis of DNA repair activity for ynk1, the yeast homologue of the metastasis suppressor NM23

4320 NM23-H1 was first identified by virtue of its reduced expression in highly metastatic melanoma and breast carcinoma cells, and the ability of forced NM23-H1 expression to inhibit metastatic potential without significant impact on the transformed phenotype. NM23-H1 exhibits nucleoside diphosphate kinase (NDPK) activity, catalyzing the transfer of γ-phosphate between nucleoside triphosphate and nucleoside diphosphate via a “ping-pong” mechanism. In addition to NDPK activity, NM23-H1 also exhibits both protein histidine kinase and 3’-5’exonuclease activities. Generally, enzymes that contain 3’-5’ exonuclease activity are involved directly in DNA repair processes and the maintenance of genomic stability. We hypothesized that loss of the 3’-5’ exonuclease activity of NM23-H1 during tumor progression impairs genomic integrity, thereby enhancing metastatic potential. In an effort to quantify the contribution of NM23-H1 to DNA repair, we have initiated experiments in the yeast Saccharomyces cerevisiae. Yeast provide excellent eukaryotic models for DNA repair research, since knockout strains are available for essentially all genes and DNA repair mechanisms are relatively well-characterized. In this project, we measured the extent to which expression of ynk1(the yeast homologue of the human metastasis suppressor NM23-H1) is required for genomic integrity. Mutation rates were measured in a haploid ynk1 knockout (ynk1Δ) strain using the standard CAN1 forward mutation assay. The ynk1 deletion had no effect on spontaneous mutation rate, but did result in increased mutations (2.2-fold) upon treatment with the DNA methylating agent, methyl methanesulfonate. Moreover, UV radiation induced a more marked (7-fold) deficit of DNA repair in the ynk1Δ strain as compared with the wild-type counterpart. Current efforts are being devoted to analyzing DNA repair activity in ynk1Δ strains that harbor second knockouts in a variety of known DNA repair genes (e.g. Ddc1, Rad17, Mec3, Msh2 and Fen1). Such double-knockout strains may magnify the ynk1-dependent mutator phenotype, as well as facilitate identification of the relevant biochemical activity(s) of the NM23-H1 molecule by complementation with wild-type and enzymatically-defective mutant forms of the protein.