Dion Vincent, Lin Yunfu, Price Brandee A, Fyffe Sharyl L, Seluanov Andrei, Gorbunova Vera, Wilson John H
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
DNA Repair (Amst). 2008 Feb 1;7(2):313-20. doi: 10.1016/j.dnarep.2007.11.002.
Trinucleotide repeat instability is intrinsic to a family of human neurodegenerative diseases. The mechanism leading to repeat length variation is unclear. We previously showed that treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) dramatically increases triplet repeat instability in mammalian cells. Based on previous reports that demethylation increases homologous recombination (HR), and our own observations that HR destabilizes triplet repeats, we hypothesized that demethylation alters repeat stability by stimulating HR. Here, we test that hypothesis at the adenosine phosphoribosyl transferase (Aprt) locus in CHO cells, where CpG demethylation and HR have both been shown to increase CAG repeat instability. We find that the rate of HR at the Aprt locus is not altered by demethylation. The spectrum of recombinants, however, was shifted from the usual 6:1 ratio of conversions to crossovers to more equal proportions in 5-aza-CdR-treated cells. The subtle influences of demethylation on HR at the Aprt locus are not sufficient to account for its dramatic effects on repeat instability. We conclude that 5-aza-CdR promotes triplet repeat instability independently of HR.
三核苷酸重复序列不稳定是一类人类神经退行性疾病所固有的特征。导致重复长度变异的机制尚不清楚。我们之前的研究表明，用去甲基化剂5-氮杂-2'-脱氧胞苷（5-aza-CdR）处理可显著增加哺乳动物细胞中的三联体重复序列不稳定性。基于之前关于去甲基化会增加同源重组（HR）的报道，以及我们自己观察到HR会使三联体重复序列不稳定，我们推测去甲基化通过刺激HR来改变重复序列的稳定性。在这里，我们在CHO细胞的腺苷磷酸核糖转移酶（Aprt）位点验证这一假设，在该位点，CpG去甲基化和HR均已被证明会增加CAG重复序列的不稳定性。我们发现，去甲基化不会改变Aprt位点的HR速率。然而，在经5-aza-CdR处理的细胞中，重组体的谱系从通常6:1的转换与交换比例转变为更接近相等的比例。去甲基化对Aprt位点HR的细微影响不足以解释其对重复序列不稳定性的显著作用。我们得出结论，5-aza-CdR促进三联体重复序列不稳定的作用独立于HR。
