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Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.

TitleMutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.
Publication TypeJournal Article
Year of Publication1995
AuthorsOnel K, Thelen MP, Ferguson DO, Bennett RL, Holloman WK
JournalMol Cell Biol
Volume15
Issue10
Pagination5329-38
Date Published1995 Oct
ISSN0270-7306
KeywordsAmino Acid Sequence, Base Sequence, DNA Repair, Escherichia coli, Exodeoxyribonuclease V, Exodeoxyribonucleases, Fungal Proteins, Genes, Fungal, Histidine, Hygromycin B, Introns, Molecular Sequence Data, Molecular Weight, Mutagenesis, Peptides, Radiation Tolerance, Recombinant Fusion Proteins, RNA Splicing, RNA, Messenger, Sequence Analysis, DNA, Sequence Deletion, Transcription, Genetic, Ustilago
Abstract

The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues. Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and production of a protein of 463 amino acid residues. The 3'-->5' exonuclease activity of proteins derived from the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated. The mutants included a series of deletions constructed by removing restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity. All of these proteins were overproduced in Escherichia coli as N-terminal polyhistidine-tagged fusions that were subsequently purified by immobilized metal affinity chromatography and assayed for 3'-->5' exonuclease activity. The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3'-->5' exonuclease activity, but deletion into the midsection caused a severe loss of activity. The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3'-->5' exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated. The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant. The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not. These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair.

Alternate JournalMol. Cell. Biol.
PubMed ID7565682
PubMed Central IDPMC230781
Grant ListGM42482 / GM / NIGMS NIH HHS / United States

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