The REC1 gene of Ustilago maydis involved in the cellular response to DNA damage encodes an exonuclease
Thelen, M.P. | Onel, K. | Holloman, W.K.
Mutation in the REC1 gene of Ustilago maydis is known to lead to a complex phenotype with alterations in DNA repair, recombination, mutagenesis, meiosis, and cell division. The predicted product of the REC1 gene is a polypeptide of 522 amino acid residues with a molecular mass of 56,866 daltons, with no overall sequence homology to any other known protein. The open reading frame of the REC1 gene placed by itself in a U. maydis expression vector was found to be sufficient to complement the reel mutant. Overexpression of REC1 in Escherichia coli gave rise to the anticipated 57-kDa product together with a 3'-5' exonuclease activity. This activity was only present in cells overexpressing REC1 and its characteristics were distinguishable from the major bacterial nucleases, but it had certain enzymatic features in common with epsilon, the proofreading exonuclease subunit of E. coli DNA polymerase III holoenzyme. To facilitate isolation of the protein product from bacteria, the REC1 gene was overexpressed from a vector that fused a hexa-histidine-leader sequence onto the amino terminus, enabling the isolation of the HisREC1 product on an immobilized metal ion affinity column. The His-REC1 protein co-eluted with the novel exonuclease activity. Alignment of the amino acid sequence of the REC1 gene product with the conserved proofreading exonuclease motifs of DNA polymerases indicated significant homology.
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