MUTATION AVOIDANCE AND DNA-REPAIR PROFICIENCY IN USTILAGO-MAYDIS ARE DIFFERENTIALLY LOST WITH PROGRESSIVE TRUNCATION OF THE REC1 GENE-PRODUCT

The REC1 gene of Ustilago maydis has an uninterrupted open reading fra me, predicted from the genomic sequence to encode a protein of 522 ami no acid residues, Nevertheless, an intron is present, and functional a ctivity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and pr oduction of a protein of 463 amino acid residues, The 3'--> 5' exonucl ease activity of proteins derived from the REC1 genomic open reading f rame, the intronless open reading frame, and several mutants was inves tigated, The mutants included a series of deletions constructed by rem oving restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation s ensitivity, All of these proteins were overproduced in Escherichia col i as N-terminal polyhistidine-tagged fusions that were subsequently pu rified by immobilized metal affinity chromatography and assayed for 3' --> t5' exonuclease activity, The results indicated that elimination o ff the C-terminal third of the protein did not result in a serious red uction in 3'--> 5' exonuclease activity, but deletion into the midsect ion caused a severe loss of activity, The biological activity of the r ecl-1 allele, which encodes a truncated polypeptide with full 3'--> 5' exonuclease activity, and the recl-5 allele, which encodes a more sev erely truncated polypeptide with no exonuclease activity, was investig ated. The two mutants were equally sensitive to the lethal effect of U V light, but the spontaneous mutation rate was elevated 10-fold over t he wild-type rate in the recl-1 mutant and 100-fold in the recl-5 muta nt. The elevated spontaneous mutation rate correlated with the ablatio n of exonuclease activity, but the radiation sensitivity did not, Thes e results indicate that the C-terminal portion of the Red protein is n ot essential for exonuclease activity but is crucial in the role of RE C1 in DNA damage repair.