Kang S, Ohshima K, Shimizu M, Amirhaeri S, Wells R D
Institute of Biosciences and Technology, Texas A & M University, Texas Medical Center, Houston 77030, USA.
J Biol Chem. 1995 Nov 10;270(45):27014-21. doi: 10.1074/jbc.270.45.27014.
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 pausing 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.
几种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增有关。对长度从17到180的CTG三联体以及长度从9到160的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、修饰的T7 DNA聚合酶（测序酶）或人DNA聚合酶β进行引物延伸时，在CTG重复序列的特定位点强烈停顿。在70℃加热可消除停顿。随着双链DNA而非单链DNA中三联体重复序列长度的增加，停顿的程度增加。停顿位点的位置由引物杂交位点与三联体重复序列起始点之间的距离决定。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，适当长度的三联体采用一种非B构象，该构象会阻碍DNA聚合酶的前进；由此产生的闲置聚合酶可能催化滑动以产生扩增序列，从而为这种非孟德尔遗传过程提供分子基础。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如强直性肌营养不良和脆性X综合征的病因有关。
