The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in vivo.
The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in vivo.
复制标题
回文和非回文序列在体外和体内阻止 DNA 合成中的作用。
DOI：
10.1016/0022-2836(84)90266-3
复制
发表时间：
1984
影响因子：
5.6
通讯作者：
DePamphilis,ML
中科院分区：
文献类型：
--
作者：
Weaver,DT;DePamphilis,ML
使用经唯一的末端标记的DNA引物退火的环状单链M13或φx174病毒DNA模板，分析了阻止哺乳动物DNA聚合酶DNA在体外合成的特定DNA序列的性质。这种方法严格定义了起始核苷酸的位置和合成的方向，并使放射性的量与新生DNA链的数量成正比，而不是与长度成正比。通过将独特的DNA限制性片段克隆到M13 DNA中，并分离含有Watson或Crick链的病毒粒子，通过带有互补序列的模板来确定阻止位点的准确核苷酸位置。结果与回文(自互补)序列、重复序列和镜像定向的重复序列的位置相关。确定了两类DNA合成停滞位点，它们结构不同，但活性相同。I类由回文序列组成，在溶液中形成稳定的发夹结构，并在两个互补模板上阻止DNA聚合酶。无论酶从哪个方向靠近，聚合酶都会准确地停在双链DNA茎的底部。II类序列由非回文序列组成，不能用二级结构或序列对称性元件解释，其互补序列不是停滞位点。根据这些数据，提出了阻止部位的大小限制、方向和一些序列特异性。通过绘制基因组中3‘端标记的新生猿猴病毒40DNA链的位置，也在活体中观察到了滞留部位。最接近复制终止区域的滞留位置最明显，在体外同一模板上，80%的最显著位置似乎被α-聚合酶识别。然而，在活体中没有发现I类位点，这表明回文序列不会在复制叉处形成发夹结构。
The nature of specific DNA sequences that arrest synthesis by mammalian DNA polymerase αin vitrowas analyzed using circular, single-stranded M13 or φX174 virion DNA templates annealed to a unique, terminally labeled, DNA primer. This method rigorously defined both the starting nucleotide position and the direction of synthesis, as well as making the amount of radioactivity proportional to the number rather than the length of nascent DNA chains. The precise nucleotide locations of arrest sites were determined over templates with complementary sequences by cloning unique DNA restriction fragments into M13 DNA and isolating virions containing either the Watson or Crick strand. Results were correlated with the locations of palindromic (self-complementary) sequences, repeated sequences, and repeated sequences with mirror-image orientation. Two classes of DNA synthesis arrest sites were identified, distinct in structure but equivalent in activity. Class I sites consisted of palindromic sequences that formed a stable hairpin structure in solution and arrested DNA polymerase on both complementary templates. The polymerase stopped precisely at the base of the duplex DNA stem, regardless of the direction from which the enzyme approached. Class II sites consisted of non-palindromic sequences that could not be explained by either secondary structure or sequence symmetry elements, and whose complementary sequence was not an arrest site. Size limits, orientation and some sequence specificity for arrest sites were suggested by the data. Arrest sites were also observedin vivoby mapping the locations of 3′-end-labeled nascent simian virus 40 DNA strands throughout the genome. Arrest sites closest to the region where termination of replication occurs were most pronounced, and the locations of 80% of the most prominent sites appeared to be recognized by α-polymerase on the same templatein vitro. However, class I sites were not identifiedin vivo, suggesting that palindromic sequences do not form hairpin structures at replication forks.