Discrimination and versatility in mismatch repair
Discrimination and versatility in mismatch repair
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DOI：
10.1016/j.dnarep.2005.09.002
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发表时间：
2005-12-08
期刊：
影响因子：
3.8
通讯作者：
Wang, HX
中科院分区：
文献类型：
--
作者：
Hays, JB;Hoffman, PD;Wang, HX
进化保守的错配修复 (MMR) 系统通过切除-再合成途径纠正 DNA 复制中的所有或几乎所有碱基错配错误，并对许多不同的 DNA 损伤做出反应。考虑到 DNA 聚合酶错误率以及体内完美配对（同源双链）DNA 过度无端切除可能产生的后果，表明 MMR 需要将同源双链 DNA 区分开三到六个数量级。然而，使用 MMR 碱基错配识别蛋白、细菌 MutS 或真核 MSH2.MSH6 (MutS α) 进行的大量结合研究通常表明，错配 DNA 和同源双链 DNA 之间的区分因子为 5-30，具体取决于结合条件、特定错配和 DNA 序列背景。因此，下游的结合后步骤必须增加 MNIR 辨别力，而不干扰识别多种碱基错配和病变所需的多功能性。我们使用复杂但高度 MMR 活性的模型系统，将人核提取物与含有特定错配和 0.15 kbp 外定义切口的质粒底物混合，来测量错配校正中最早可量化的关键步骤，即切口处错配引发的 3'-5' 切除的启动。我们将这些结果与纯化的 MutS α 与在相同序列背景中包含相同错配的合成寡双链体的结合进行比较，条件与核提取物中普遍存在的条件非常相似。对同源双链 DNA 的歧视，在结合研究中仅为 2 至 5 倍，而在切除起始时则增加至 60 至 230 倍或更多，具体取决于特定的错配。值得注意的是，切除起始的错配偏好顺序与 hMutS α 结合的顺序发生了显着改变。这表明结合后步骤不仅强烈歧视同源双链 DNA，而且其机制不受初始结合偏好的严格限制。由合成寡聚体制备或从质粒中切出的同源双链体对（40、50和70 bp）显示出几乎相同的hMutS α结合亲和力，这表明hMutS α与同源双链体DNA的高结合不是化学合成过程中引入的错误掺入或损伤的结果。 MutS 同源物对完美配对 DNA 的内在亲和力可能有助于这些蛋白质有效地定位自己，以在 MMR 途径中执行后续的错配特异性步骤。 (c) 2005 Elsevier B.V. 保留所有权利。
Evolutionarily-conserved mismatch-repair (MMR) systems correct all or almost all base-mismatch errors from DNA replication via excision-resynthesis pathways, and respond to many different DNA lesions. Consideration of DNA polymerase error rates and possible consequences of excess gratuitous excision of perfectly paired (homoduplex) DNA in vivo suggests that MMR needs to discriminate against homoduplex DNA by three to six orders of magnitude. However, numerous binding studies using MMR base-mispair-recognition proteins, bacterial MutS or eukaryotic MSH2.MSH6 (MutS alpha), have typically shown discrimination factors between mismatched and homoduplex DNA to be 5-30, depending on the binding conditions, the particular mismatches, and the DNA-sequence contexts. Thus, downstream post-binding steps must increase MNIR discrimination without interfering with the versatility needed to recognize a large variety of base-mismatches and lesions. We use a complex but highly MMR-active model system, human nuclear extracts mixed with plasmid substrates containing specific mismatches and defined nicks 0.15 kbp away, to measure the earliest quantifiable committed step in mismatch correction, initiation of mismatch-provoked 3'-5' excision at the nicks. We compared these results to binding of purified MutS alpha to synthetic oligoduplexes containing the same mismatches in the same sequence Contexts, under conditions very similar to those prevailing in the nuclear extracts. Discrimination against homoduplex DNA, only two-to five-fold in the binding studies, increased to 60- to 230-fold or more for excision initiation, depending on the particular mismatches. Remarkably, the mismatch-preference order for excision initiation was substantially altered from the order for hMutS alpha binding. This suggests that post-binding steps not only strongly discriminate against homoduplex DNA, but do so by mechanisms not tightly constrained by initial binding preferences. Pairs of homoduplexes (40, 50, and 70 bp) prepared from synthetic oligomers or cut out of plasmids showed virtually identical hMutS alpha binding affinities, suggesting that high hMutS alpha binding to homoduplex DNA is not the result of misincorporations or lesions introduced during chemical synthesis. Intrinsic affinities of MutS homologs for perfectly paired DNA may help these proteins efficiently position themselves to carry out subsequent mismatch-specific steps in MMR pathways. (c) 2005 Elsevier B.V. All rights reserved.