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首页> 外文期刊>Molecular and Cellular Biology >Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.
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Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.

机译：随着REC1基因产物的逐步截短，Ustilago maydis中的避免突变和DNA修复能力会有所不同。

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摘要

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.
机译：从基因组序列预测，Ustilago maydis的REC1基因具有不间断的开放阅读框，可编码522个氨基酸残基的蛋白质。然而，存在内含子，并且该基因在有丝分裂细胞中的功能活性需要RNA加工事件以除去内含子。这导致阅读框的改变和463个氨基酸残基的蛋白质的产生。研究了从REC1基因组开放阅读框，无内含子开放阅读框和几个突变体衍生的蛋白质的3'-> 5'核酸外切酶活性。突变体包括一系列的缺失，这些缺失是通过去除克隆的REC1基因3'末端的限制性片段而构建的，以及一系列先前在放射敏感性筛选中分离的突变等位基因。所有这些蛋白质在大肠杆菌中均以N末端多组氨酸标记的融合蛋白过量生产，随后通过固定的金属亲和色谱纯化，并检测3'-> 5'核酸外切酶活性。结果表明，消除蛋白质的C末端三分之一不会导致3'-> 5'核酸外切酶活性的严重降低，但是缺失到中部会导致活性的严重丧失。研究了rec1-1等位基因的生物学活性，该基因编码具有完整3'-> 5'核酸外切酶活性的截短的多肽，而rec1-5等位基因，其编码具有更严格的截短而没有核酸外切酶活性的多肽。这两个突变体对紫外线的致死作用同样敏感，但是自发突变率在rec1-1突变体中比野生型率提高了10倍，在rec1-5突变体中提高了100倍。自发突变率的升高与核酸外切酶活性的降低相关，但辐射敏感性却没有。这些结果表明，Rec1蛋白的C端部分对于核酸外切酶活性不是必需的，但对REC1在DNA损伤修复中的作用至关重要。

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