PAUSING OF DNA-SYNTHESIS IN-VITRO AT SPECIFIC LOCI IN CTG AND CGG TRIPLET REPEATS FROM HUMAN HEREDITARY-DISEASE GENES
PAUSING OF DNA-SYNTHESIS IN-VITRO AT SPECIFIC LOCI IN CTG AND CGG TRIPLET REPEATS FROM HUMAN HEREDITARY-DISEASE GENES
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DOI：
10.1074/jbc.270.45.27014
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发表时间：
1995-11-10
影响因子：
4.8
通讯作者：
WELLS, RD
中科院分区：
文献类型：
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作者：
KANG, SM;OHSHIMA, K;WELLS, RD
几个人类遗传性神经肌肉疾病基因与CTG或CGG三联体重复序列的扩大有关。对17~180个CTG三联体和9~160个CGG重复序列的DNA合成进行了体外研究。使用DNA聚合酶I的Klenow片段、改良的T7 DNA聚合酶(Sequenase)或人DNA聚合酶β的引物延伸在CTG重复序列中的特定位置强烈停顿。随着双链DNA中三联体重复序列的长度增加，而单链DNA中的三联体重复序列的长度增加，停顿的幅度增加。间歇点的位置由引物杂交位点到三联体重复序列开始之间的距离来决定。CGG三联体重复序列也显示出相似但不完全相同的停顿模式。这些结果表明，三联体的适当长度采用非B构象(S)，阻止了DNA聚合酶的进展；所产生的空闲聚合酶可能催化滑移而得到扩展的序列，从而为这种非孟德尔遗传过程提供了分子基础。如果这些机制存在于人类细胞中，可能与某些神经肌肉疾病的病因有关，如强直性肌营养不良和脆性X综合征。
Several human hereditary neuromuscular disease genes are associated with the expansion of CTG or CGG triplet repeats. The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase beta paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70 degrees C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausings increased. The location of the pause sites was determined by the distance between the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for this non-Mendelian genetic process. These mechanisms, if present in human cells, may be related to the etiology of certain neuromuscular diseases such as myotonic dystrophy and Fragile X syndrome.