Liu Guoqi, Bissler John J, Sinden Richard R, Leffak Michael
Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435, USA.
Mol Cell Biol. 2007 Nov;27(22):7828-38. doi: 10.1128/MCB.01276-07. Epub 2007 Sep 10.
Spinocerebellar ataxia type 10 (SCA10) is associated with expansion of (ATTCT)n repeats (where n is the number of repeats) within the ataxin 10 (ATX10/E46L) gene. The demonstration that (ATTCT)n tracts can act as DNA unwinding elements (DUEs) in vitro has suggested that aberrant replication origin activity occurs at expanded (ATTCT)n tracts and may lead to their instability. Here, we confirm these predictions. The wild-type ATX10 locus displays inefficient origin activity, but origin activity is elevated at the expanded ATX10 loci in patient-derived cells. To test whether (ATTCT)n tracts can potentiate origin activity, cell lines were constructed that contain ectopic copies of the c-myc replicator in which the essential DUE was replaced by ATX10 DUEs with (ATTCT)n. ATX10 DUEs containing (ATTCT)27 or (ATTCT)48, but not (ATTCT)8 or (ATTCT)13, could substitute functionally for the c-myc DUE, but (ATTCT)48 could not act as an autonomous replicator. Significantly, chimeric c-myc replicators containing ATX10 DUEs displayed length-dependent (ATTCT)n instability. By 250 population doublings, dramatic two- and fourfold length expansions were observed for (ATTCT)27 and (ATTCT)48 but not for (ATTCT)8 or (ATTCT)13. These results implicate replication origin activity as one molecular mechanism associated with the instability of (ATTCT)n tracts that are longer than normal length.
10型脊髓小脑共济失调（SCA10）与共济失调10（ATX10/E46L）基因内（ATTCT）n重复序列（其中n为重复次数）的扩增有关。（ATTCT）n序列在体外可作为DNA解旋元件（DUEs）的证据表明，异常的复制起始点活性发生在扩增的（ATTCT）n序列处，并可能导致其不稳定。在此，我们证实了这些预测。野生型ATX10基因座显示出低效的起始点活性，但在患者来源细胞中扩增的ATX10基因座处起始点活性升高。为了测试（ATTCT）n序列是否能增强起始点活性，构建了含有c-myc复制子异位拷贝的细胞系，其中必需的DUE被带有（ATTCT）n的ATX10 DUE取代。含有（ATTCT）27或（ATTCT）48但不含有（ATTCT）8或（ATTCT）13的ATX10 DUE能够在功能上替代c-myc DUE，但（ATTCT）48不能作为自主复制子。值得注意的是，含有ATX10 DUE的嵌合c-myc复制子表现出长度依赖性的（ATTCT）n不稳定性。到250次群体倍增时，观察到（ATTCT）27和（ATTCT）48有显著的两倍和四倍长度扩增，但（ATTCT）8或（ATTCT）13没有。这些结果表明复制起始点活性是与长于正常长度的（ATTCT）n序列不稳定性相关的一种分子机制。
