Morag A S, Saveson C J, Lovett S T
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, 02454-9110, USA.
J Mol Biol. 1999 May 28;289(1):21-7. doi: 10.1006/jmbi.1999.2763.
Duplication or expansion of directly repeated sequence elements is associated with a number of human genetic diseases. To study the mechanisms of repeat expansion, we have developed a plasmid assay in Escherichia coli. Our assay involves two simple repeats of 787 bp in length; expansion to three or more copies of the repeat can be selected by restoration of an intact tetracycline-resistance gene. Expansions occurred at relatively high rates, >10(-5), in the population. Both RecA-dependent recombination and RecA-independent slipped misalignments contributed to the observed expansion events. Mutations that impair DNA polymerase III (DnaE, DnaQ subunits) or the replication fork helicase, DnaB, stimulated both RecA-dependent and RecA-independent expansion events. In these respects, the properties of repeat expansion resemble repeat deletion and suggest that difficulties in DNA replication may trigger both classes of rearrangements. About 20% of the RecA-independent expansion events are accompanied by reciprocal sister-chromosome exchange, producing dimeric plasmids carrying one triplicated and one deleted locus. These products are explained by a model involving misaligned strands across the replication fork. This model predicts that the location of a replication stall site may govern the types of resulting rearrangements. The specific location of such a stall site can also, in theory, account for propensity towards expansion or deletion of repeat arrays. This may have relevance to trinucleotide repeat expansion in human genetic disease.
直接重复序列元件的复制或扩增与多种人类遗传疾病相关。为了研究重复序列扩增的机制，我们在大肠杆菌中开发了一种质粒检测方法。我们的检测方法涉及两个长度为787 bp的简单重复序列；通过恢复完整的四环素抗性基因，可以筛选出重复序列扩增至三个或更多拷贝的情况。在群体中，扩增以相对较高的频率发生，>10(-5)。RecA依赖性重组和RecA非依赖性滑动错配都导致了观察到的扩增事件。损害DNA聚合酶III（DnaE、DnaQ亚基）或复制叉解旋酶DnaB的突变，刺激了RecA依赖性和RecA非依赖性扩增事件。在这些方面，重复序列扩增的特性类似于重复序列缺失，表明DNA复制中的困难可能引发这两类重排。约20%的RecA非依赖性扩增事件伴随着相互的姐妹染色单体交换，产生携带一个三倍体和一个缺失位点的二聚体质粒。这些产物可以用一个涉及复制叉处错配链的模型来解释。该模型预测，复制停滞位点的位置可能决定所产生的重排类型。理论上，这样一个停滞位点的特定位置也可以解释重复序列阵列扩增或缺失的倾向。这可能与人类遗传疾病中的三核苷酸重复扩增有关。
