Streisinger revisited: DNA synthesis errors mediated by substrate misalignments
Streisinger revisited: DNA synthesis errors mediated by substrate misalignments
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DOI：
10.1101/sqb.2000.65.81
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发表时间：
2000-01-01
期刊：
影响因子：
--
通讯作者：
Kunkel, TA
中科院分区：
文献类型：
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作者：
Bebenek, K;Kunkel, TA
《冷泉港定量生物学研讨会》，卷LXV，© 2000冷泉港实验室出版社0-87969-605-2/00。81形式;为了简单起见，对于每个游程长度仅示出一个中间体。此外，重复序列越长，引物末端和双链体中额外核苷酸之间的距离越大，潜在地减少DNA聚合酶对后续掺入事件的干扰。支持这些概念的是观察到DNA聚合酶移码错误率随着均聚运行长度的增加而增加。例如，人DNA聚合酶-β（pol-β）的缺失错误率（图2b中的灰色条）和核酸外切酶缺陷型T7 DNA聚合酶的添加错误率（图2b中的黑色条）都随着连续胸苷数目的增加而增加。其他几种DNA聚合酶的合成也有类似的相关性。与这种关系一致的事实是，通过缩短或中断运行，可以降低或消除pol-β（Kunkel 1985 b）和人类免疫缺陷病毒1型逆转录酶（HIV-1 RT）（Bebenek et al. 1993）单碱基缺失的均聚模板热点处的错误率。Streisinger的逻辑也得到了DNA聚合酶的结构-功能研究的支持，这在1966年是不可能的。例如，1998年描述的与DNA结合的T7 DNA聚合酶的晶体结构（Doublié et al. 1998）揭示了与模板-引物接触的许多氨基酸，这些氨基酸在聚合酶活性位点上游的几个碱基对处发生（图2c）。这些包括与DNA骨架和小沟的相互作用，当聚合酶结合时，小沟比典型的B-DNA更宽和更浅（图2d）。一种变异的衍生物-
Cold Spring Harbor Symposia on Quantitative Biology, Volume LXV.© 2000 Cold Spring Harbor Laboratory Press 0-87969-605-2/00. 81 form; for simplicity, only one intermediate is shown for each run length. In addition, the longer the repetitive sequence, the greater the distance between the primer terminus and the extra nucleotide in the duplex, potentially reducing interference in subsequent incorporation events by a DNA polymerase. In favor of these concepts is the observation that DNA polymerase frameshift error rates increase as the length of a homopolymeric run increases. For example, the deletion error rate of human DNA polymerase-β (pol-β)(gray bars in Fig. 2b) and the addition error rate of exonuclease-deficient T7 DNA polymerase (black bars in Fig. 2b) both increase as the number of consecutive thymidines increases. Similar correlations have been made for synthesis by several other DNA polymerases. Also consistent with this relationship is the fact that error rates at homopolymeric template hot spots for single-base deletions by pol-β (Kunkel 1985b) and human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT)(Bebenek et al. 1993) can be reduced or eliminated by shortening or interrupting the run. Streisinger’s logic is also supported by structure-function studies of DNA polymerases that were not possible in 1966. For example, the crystal structure of T7 DNA polymerase bound to DNA described in 1998 (Doublié et al. 1998) revealed numerous amino acid contacts with the template-primer that occur several base pairs upstream of the polymerase active site (Fig. 2c). These include interactions with the DNA backbone and in the minor groove, which is wider and shallower than is typical of B-DNA when the polymerase is bound (Fig. 2d). A mutant deriva-