Leonard Jeffrey M, Bollmann Stephanie R, Hays John B
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331-7301, USA.
Plant Physiol. 2003 Sep;133(1):328-38. doi: 10.1104/pp.103.023952.
Highly conserved mismatch repair (MMR) systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination, and responding to various DNA lesions. Arabidopsis and other plants encode a suite of MMR protein orthologs, including MSH2, the constant component of various specialized eukaryotic mismatch recognition heterodimers. To study MMR roles in plant genomic stability, we used Arabidopsis AtMSH2::TDNA mutant SALK_002708 and AtMSH2 RNA-interference (RNAi) lines. AtMSH2::TDNA and RNAi lines show normal growth, development, and fertility. To analyze AtMSH2 effects on germ line DNA fidelity, we measured insertion-deletion mutation of dinucleotide-repeat sequences (microsatellite instability) at nine loci in 16 or more progeny of two to four different wild-type or AtMSH2-deficient plants. Scoring 992 total alleles revealed 23 (2.3%) unique and 51 (5.1%) total repeat length shifts ([+2], [-2], [+4], or [-4] bp). For the six longest repeat loci, the corresponding frequencies were 22/608 and 50/608. Two of four AtMSH2-RNAi plants showed similar microsatellite instability. In wild-type progeny, only one unique repeat length allele was found in 576 alleles tested. This endogenous microsatellite instability, shown for the first time in MMR-defective plants, is similar to that seen in MMR-defective yeast and mice, indicating that plants also use MMR to promote germ line fidelity. We used a frameshifted reporter transgene, (G)(7)GUS, to measure insertion-deletion reversion as blue-staining beta-glucuronidase-positive leaf spots. Reversion rates increased only 5-fold in AtMSH2::TDNA plants, considerably less than increases in MSH2-deficient yeast or mammalian cells for similar mononucleotide repeats. Thus, MMR-dependent error correction may be less stringent in differentiated leaf cells than in plant equivalents of germ line tissue.
高度保守的错配修复（MMR）系统通过校正DNA复制错误、对抗同源重组以及对各种DNA损伤作出反应来促进基因组稳定性。拟南芥和其他植物编码一套MMR蛋白直系同源物，包括MSH2，它是各种特殊真核错配识别异二聚体的恒定成分。为了研究MMR在植物基因组稳定性中的作用，我们使用了拟南芥AtMSH2::TDNA突变体SALK_002708和AtMSH2 RNA干扰（RNAi）株系。AtMSH2::TDNA和RNAi株系表现出正常的生长、发育和育性。为了分析AtMSH2对生殖系DNA保真度的影响，我们在两到四株不同野生型或AtMSH2缺陷型植物的16个或更多后代中的九个位点测量了二核苷酸重复序列的插入缺失突变（微卫星不稳定性）。对总共992个等位基因进行评分，发现有23个（2.3%）独特的和51个（5.1%）总的重复长度变化（[+2]、[-2]、[+4]或[-4] bp）。对于六个最长的重复位点，相应的频率分别为22/608和50/608。四株AtMSH2-RNAi植物中有两株表现出相似的微卫星不稳定性。在野生型后代中，在测试的576个等位基因中仅发现一个独特的重复长度等位基因。这种内源性微卫星不稳定性首次在MMR缺陷型植物中被观察到，类似于在MMR缺陷型酵母和小鼠中看到的情况，表明植物也利用MMR来促进生殖系保真度。我们使用了一个移码报告转基因（G）（7）GUS，通过蓝色染色的β-葡萄糖醛酸酶阳性叶斑来测量插入缺失回复。在AtMSH2::TDNA植物中回复率仅增加了5倍，远低于MSH2缺陷型酵母或哺乳动物细胞中类似单核苷酸重复的增加幅度。因此，MMR依赖的错误校正在分化的叶细胞中可能不如在植物生殖系组织等同物中严格。
