Isolation of crt mutants constitutive for transcription of the DNA damage inducible gene RNR3 in Saccharomyces cerevisiae

Abstract

Ribonucleotide reductase is an essential enzyme that catalyzes the rate limiting step for production of the deoxyribonucleotides required for DNA synthesis. It is encoded by three genes, RNR1, RNR2 and RNR3, each of which is inducible by agents that damage DNA or block DNA replication. To probe the signaling pathway mediating this DNA damage response, we have designed a general selection system for isolating spontaneous trans-acting mutations that alter RNR3 expression using a chromosomal RNR3-URA3 transcriptional fusion and an RNR3-lacZ reporter plasmid. Using this system, we have isolated 202 independent trans-acting crt (constitutive RNR3 transcription) mutants that express high levels of RNR3 in the absence of DNA damaging agents. Of these, 200 are recessive and fall into 9 complementation groups. In some crt groups, the expression of RNR1 and RNR2 are also elevated, suggesting that all three RNR genes share a common regulatory pathway. Mutations in most CRT genes confer additional phenotypes, among these are clumpiness, hydroxyurea sensitivity, temperature sensitivity and slow growth. Five of the CRT genes have been identified as previously cloned genes; CRT4 is TUP1, CRT5 is POL1/CDC17, CRT6 is RNR2, CRT7 is RNR1, and CRT8 is SSN6. crt6-68 and crt7- 240 are the first ts alleles of RNR2 and RNR1, respectively, and arrest with a large budded, cdc terminal phenotype at the nonpermissive temperature. The isolation of crt5-262, an additional cdc allele of POL1/CDC17, suggests for the first time that directly blocking DNA replication can provide a signal to induce the DNA damage response. crt2 mutants show a defect in basal level expression of RNR1-lacZ reporter constructs. These are the first mutants isolated in yeast that alter the regulation of DNA damage inducible genes and the identification of their functions sheds light on the DNA damage sensory network.