Pollard Laura M, Sharma Rajesh, Gómez Mariluz, Shah Sonali, Delatycki Martin B, Pianese Luigi, Monticelli Antonella, Keats Bronya J B, Bidichandani Sanjay I
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Nucleic Acids Res. 2004 Nov 8;32(19):5962-71. doi: 10.1093/nar/gkh933. Print 2004.
Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA*TTC)n sequences (9-105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8-107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.
弗里德赖希共济失调是由FRDA基因中多态性且不稳定的GAA三联体重复序列扩增所致，但其不稳定的机制尚不清楚。在大肠杆菌中繁殖的质粒中，(GAA*TTC)n序列（9 - 105个三联体）的复制表现出长度和方向依赖性的不稳定性。两个方向复制时都有小的长度变化，但当GAA作为后随链模板时，经常观察到大幅收缩。这个方向的DNA复制也明显更慢。为了评估我们研究结果的生理相关性，我们分析了携带相似长度重复序列（8 - 107个三联体）的人类受试者的外周血白细胞。使用小池PCR分析9400个体细胞FRDA分子，发现了类似的突变谱，包括大幅收缩。体内体细胞不稳定性起始的阈值长度在40至44个三联体之间，与真核冈崎片段的长度相对应。与生殖系传递过程中前突变等位基因的稳定一致，我们还发现(GAGGAA)n六核苷酸插入消除了体内体细胞和在大肠杆菌中繁殖的重复序列的不稳定性。我们的数据表明，弗里德赖希共济失调中的GAA三联体重复突变在DNA复制过程中不稳定，经常发生大幅收缩。
