A DNA polymerase alpha pause site is a hot spot for nucleotide misinsertion.
A DNA polymerase alpha pause site is a hot spot for nucleotide misinsertion.
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DNA 聚合酶 α 暂停位点是核苷酸错误插入的热点。
DOI：
10.1073/pnas.89.2.763
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发表时间：
1992
影响因子：
11.1
通讯作者：
Loeb,LA
中科院分区：
文献类型：
--
作者：
Fry,M;Loeb,LA
在这项研究中，我们检查了 DNA 聚合酶 α (pol α) 催化的 DNA 合成在模板暂停位点的停滞是否会导致末端核苷酸错误掺入。开发了一种方法来识别新生 DNA 链中在暂停位点积累的 3' 末端核苷酸。将放射性 5' 端标记引物与噬菌体 M13mp2 单链 DNA 模板退火，并通过 pol α 进行延伸。通过测序凝胶电泳解析、分离和纯化在暂停位点积累的单个DNA链。这些 DNA 链通过 pol α 使用四个退火的合成 DNA 模板进行延长，每个模板在与捕获链 3' 末端相对的位置包含不同的核苷酸。由于 pol α 对匹配引物末端的高度偏好，因此只有那些含有与分离的暂停位点链 3' 末端互补的核苷酸的模板才会被复制。产物 DNA 的电泳显示了每个模板的复制程度，从而鉴定了暂停位点链的 3' 末端核苷酸。我们发现产物DNA链以与M13mp2基因组6272-6269位置处的序列3'-d(AAAA)-5'内的暂停位点相对的非互补3'端核苷酸终止。 pol α 催化 dG 或 dA 错误掺入新生链的 3' 末端，与 M13mp2 模板 dA 残基的两个相对。对不同暂停位点的类似分析并未揭示与模板 dC 相对的显着错误掺入。这些结果表明，一些但不是所有 pol α 暂停的位点可能构成诱变位点。
In this study we examined whether the arrest of DNA polymerase alpha (pol alpha)-catalyzed DNA synthesis at template pause sites entails terminal nucleotide misincorporation. An approach was developed to identify the 3'-terminal nucleotide in nascent DNA chains that accumulate at pause sites. A radioactive 5'-end-labeled primer was annealed to a bacteriophage M13mp2 single-stranded DNA template and elongated by pol alpha. Individual DNA chains that were accumulated at pause sites were resolved by sequencing gel electrophoresis, isolated, and purified. These DNA chains were elongated by pol alpha by using four annealed synthetic DNA templates, each of which contained a different nucleotide at the position opposite the 3' terminus of the arrested chain. Owing to the high preference of pol alpha for matched-over-mismatched primer termini, only those templates that contain a nucleotide that is complementary to the 3' terminus of the isolated pause-site chain are copied. Electrophoresis of product DNA showed the extent of copying of each template and thus identified the 3'-terminal nucleotide of the pause-site chains. We found that product DNA chains terminate with a noncomplementary 3'-terminal nucleotide opposite pause sites within the sequence 3'-d(AAAA)-5' at positions 6272-6269 of the M13mp2 genome. pol alpha catalyzed misincorporation of dG or dA into the 3' terminus of nascent chains opposite two of the M13mp2 template dA residues. A similar analysis of a different pause site did not reveal significant misincorporation opposite template dC. These results suggest that some but not all sites at which pol alpha pauses may constitute loci of mutagenesis.