THE SCHIZOSACCHAROMYCES-POMBE RAD3 CHECKPOINT GENE

The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conse rved protein which is required for all the DNA structure checkpoints i n both yeast model systems. Rad3 is an inessential member of the 'lipi d kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitat ion of overexpressed Rad3 demonstrates an associated protein kinase ac tivity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that R ad3 exists in a complex containing multiple copies of Rad3. We have id entified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation se nsitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underl ying the DNA structure checkpoints are conserved throughout evolution.