Trinucleotide repeat expansions catalyzed by human cell-free extracts.
Trinucleotide repeat expansions catalyzed by human cell-free extracts.
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作者：
三核苷酸重复序列的扩张导致了17种可遗传的人类神经疾病。在一些疾病中，躯体扩张发生在非增殖性组织中，如DNA复制受限的大脑。这一发现激发了人们对复制无关的扩展机制的浓厚兴趣。异常的dna修复可能是一个来源，部分基于小鼠研究表明，dna修复蛋白mutsβ(msh2-msh3复合体)刺激了体细胞的扩张。到目前为止，生化研究使用了无细胞提取物或纯化的DNA修复蛋白来产生三联体重复的部分反应。这些发现包括在一条链上但不在另一条链上扩张，或者加工被认为是扩张过程中重要中间体的DNA发夹结构。然而，很难概括完整的体外扩增过程，而且MutSβ的生化作用仍然存在争议。在这里，我们使用一种新的体外试验来证明，人类无细胞提取物催化三核苷酸重复序列的扩张和收缩，而不需要DNA复制。这种提取物促进了一系列类似于某些疾病的扩张，三联体重复长度和序列控制着体外和体内的扩张。MutsDNA刺激提取物中的扩张，与作为扩张来源的内源性β损伤的异常修复一致。总体而言，这一生化系统保留了人类和小鼠体细胞扩张的关键特征，表明这一重要的突变过程可以在试管中恢复。
Trinucleotide repeat expansions cause 17 heritable human neurological disorders. In some diseases, somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited. This finding stimulated significant interest in replication-independent expansion mechanisms. Aberrant DNA repair is a likely source, based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutSβ (Msh2-Msh3 complex). Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats. The findings included expansions on one strand but not the other, or processing of DNA hairpin structures thought to be important intermediates in the expansion process. However, it has been difficult to recapitulate complete expansions in vitro, and the biochemical role of MutSβ remains controversial. Here, we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication. The extract promotes a size range of expansions that is similar to certain diseases, and triplet repeat length and sequence govern expansions in vitro as in vivo. MutSβ stimulates expansions in the extract, consistent with aberrant repair of endogenous DNA damage as a source of expansions. Overall, this biochemical system retains the key characteristics of somatic expansions in humans and mice, suggesting that this important mutagenic process can be restored in the test tube.
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影响因子：
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DOI：
10.1073/pnas.1105461108
发表时间：
2011-10-18
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