Ohshima K, Wells R D
Center for Genome Research, Institute of Biosciences and Technology, Texas A & M University, Texas Medical Center, 2121 W. Holcombe Blvd., Houston, Texas 77030-3303, USA.
J Biol Chem. 1997 Jul 4;272(27):16798-806. doi: 10.1074/jbc.272.27.16798.
Genetic expansion of DNA triplet repeat sequences (TRS) found in neurogenetic disorders may be due to abnormal DNA replication. We have previously observed strong DNA synthesis pausings at specific loci within the long tracts (> approximately 70 repeats) of CTG.CAG and CGG.CCG as well as GTC.GAC by primer extensions in vitro using DNA polymerases (the Klenow fragment of Escherichia coli DNA polymerase I, the modified T7 DNA polymerase (Sequenase), and human DNA polymerase beta). Herein, we have isolated and analyzed the products of stalled synthesis found at approximately 30-40 triplets from the beginning of the TRS. DNA sequence analyses revealed that the stalled products contained short tracts of homogeneous TRS (6-12 repeats) in the middle of the sequence corresponding to the flanking region of the primer-template sequence. The sequence at the 3'-side terminated at the end of the primer, indicating that the primer molecule had served as a template. In addition, chemical probe and polyacrylamide gel electrophoretic analyses revealed that the stalled products existed in hairpin structures. We postulate that these products of the DNA polymerases are caused by the existence of an unusual DNA conformation(s) within the TRS, during the in vitro DNA synthesis, enhancing the DNA slippages and the hairpin formations in the TRS due to primer realignment. The consequence of these steps is DNA synthesis to the end of the primer and termination. Primer realignment including hairpin formation may play an important intermediate role in the replication of TRS in vivo to elicit genetic expansions.
在神经遗传疾病中发现的DNA三联体重复序列（TRS）的基因扩增可能是由于异常的DNA复制所致。我们之前通过体外使用DNA聚合酶（大肠杆菌DNA聚合酶I的Klenow片段、修饰的T7 DNA聚合酶（测序酶）和人DNA聚合酶β）进行引物延伸，观察到在CTG.CAG、CGG.CCG以及GTC.GAC的长片段（>约70个重复）内的特定位点存在强烈的DNA合成停顿。在此，我们分离并分析了从TRS起始处约30 - 4个三联体处发现的合成停顿产物。DNA序列分析表明，停顿产物在对应引物 - 模板序列侧翼区域的序列中间包含短的均匀TRS片段（6 - 12个重复）。3'端的序列在引物末端终止，表明引物分子充当了模板。此外，化学探针和聚丙烯酰胺凝胶电泳分析表明，停顿产物以发夹结构存在。我们推测，在体外DNA合成过程中，这些DNA聚合酶产物是由TRS内存在异常DNA构象引起的，由于引物重新排列，增强了TRS中的DNA滑动和发夹形成。这些步骤的结果是DNA合成到引物末端并终止。包括发夹形成在内的引物重新排列可能在体内TRS复制引发基因扩增过程中起重要的中间作用。
