Pearson C E, Ewel A, Acharya S, Fishel R A, Sinden R R
Center for Genome Research, Institute of Biosciences and Technology in the Texas Medical Center, Texas A&M University, Houston 77030, USA.
Hum Mol Genet. 1997 Jul;6(7):1117-23. doi: 10.1093/hmg/6.7.1117.
The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n x (CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure-specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.
三核苷酸重复序列的扩增与多种神经退行性疾病相关。这种扩增的机制尚不清楚，但可能涉及滑链结构，即三核苷酸重复序列中相邻而非完美互补的序列配对。在此，我们研究了人类错配修复蛋白MSH2与由三联体重复序列形成的滑链结构之间的相互作用，以探讨MSH2在三核苷酸扩增中可能的作用。我们检测了包含与疾病相关长度的(CTG)n×(CAG)n三联体重复的强直性肌营养不良基因座的基因组克隆。我们通过退火包含以下内容的DNA互补链构建了两种类型的滑链结构：(i) 等量的三核苷酸重复序列（同型双链滑链结构或S-DNA）或 (ii) 不同数量的重复序列（异型双链滑链中间体或SI-DNA）。具有过量CTG或CAG重复序列的SI-DNA在结构上是不同的，可以通过电泳分离并单独研究。使用凝胶迁移实验表明，MSH2以结构特异性方式与S-DNA和SI-DNA结合。MSH2的亲和力随着重复序列长度的增加而增加。此外，MSH2优先结合成环的CAG重复序列，这表明MSH2识别中存在链不对称性。我们的结果与MSH2可能通过其在修复和/或重组中的作用参与三核苷酸重复扩增的观点一致。
