遗传 ›› 2017, Vol. 39 ›› Issue (9): 771-774.doi: 10.16288/j.yczz.17-251

• 前沿聚焦 •    下一篇

DNA复制研究步入单分子时代

刘晓晶(),楼慧强()   

  1. 中国农业大学生物学院,农业生物技术国家重点实验室,北京 100193
  • 收稿日期:2017-07-27 修回日期:2017-08-24 出版日期:2017-09-20 发布日期:2017-10-21
  • 基金资助:
    国家自然科学基金项目(31630005)

Single molecular biology: coming of age in DNA replication

Xiaojing Liu(),Huiqiang Lou()   

  1. State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
  • Received:2017-07-27 Revised:2017-08-24 Online:2017-09-20 Published:2017-10-21
  • Supported by:
    Supported by the National Natural Foundation of China(31630005)

摘要:

DNA复制是生命体内必不可少的基本过程之一。传统研究显示DNA复制体中前导链和后随链的合成速度总体来说是一致的,从而避免在新生链中产生明显的单链缺口。主流的观点认为这是由于负责前导链和后随链的两个DNA聚合酶分子之间存在着某种协调同步机制。然而,Kowalczykowski实验室最近采用单分子荧光显微技术实时跟踪发现,大肠杆菌DNA复制体前导链和后随链上两个DNA聚合酶分子互相独立工作,并且都不是匀速行进而是呈现断断续续、时快时慢的随机动态变化。当DNA聚合酶暂停复制时,解旋酶仍会持续解链,导致解旋酶和聚合酶短暂的分离。有意思的是,此时DNA复制体触发一种类似“死人键”(dead-man’s switch)的保险机制,使DNA解旋的速度降低80%,从而恢复解旋酶和聚合酶的偶联。基于单分子水平的实时观察,他们认为前导链和后随链DNA复制进程均遵循一个符合高斯分布的随机模型。这与传统的生化研究观察到两者的合成速度总体来说是一致的并不矛盾。Kowalczykowski实验室的研究实现了从复制开始到结束整个过程对每个单分子行为的连续观测,而传统研究反映的则是经过较长时间对多分子群体平均水平的最终结果进行测定。因此,单分子技术可以极大地弥补传统生化研究的不足。随着未来单分子技术的进步和更广泛的应用,必将把包括DNA复制在内的生物学研究带到一个新的时代。

关键词: DNA复制, DNA聚合酶, 单分子技术, 随机性, 协同性

Abstract:

DNA replication is an essential process of the living organisms. To achieve precise and reliable replication, DNA polymerases play a central role in DNA synthesis. Previous investigations have shown that the average rates of DNA synthesis on the leading and lagging strands in a replisome must be similar to avoid the formation of significant gaps in the nascent strands. The underlying mechanism has been assumed to be coordination between leading- and lagging-strand polymerases. However, Kowalczykowski’s lab members recently performed single molecule techniques in E. coli and showed the real-time behavior of a replisome. The leading- and lagging-strand polymerases function stochastically and independently. Furthermore, when a DNA polymerase is paused, the helicase slows down in a self-regulating fail-safe mechanism, akin to a ‘‘dead-man’s switch’’. Based on the real-time single-molecular observation, the authors propose that leading- and lagging-strand polymerases synthesize DNA stochastically within a Gaussian distribution. Along with the development and application of single-molecule techniques, we will witness a new age of DNA replication and other biological researches.

Key words: DNA replication, DNA polymerases, single-molecule techniques, stochastic, coordination