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中国物理学会期刊

DNA双链退火压力对DNA聚合酶gp5链置换的调控

CSTR: 32037.14.aps.70.20210707

Control of DNA polymerase gp5 chain substitution by DNA double strand annealing pressure

CSTR: 32037.14.aps.70.20210707
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  • DNA聚合酶是执行DNA复制和修复的重要蛋白, 由于其只能从5′向3′方向聚合, 所以在聚合双链DNA时会有两种模式: 其一是先打开DNA双链, 让其暴露出3′-5′方向的模板链(先导链), 然后沿着这条链复制出新链以此置换旧链, 这就是链置换的合成. 另一种是沿着已经置换出的5′-3′方向的模板链(滞后链)进行延伸合成. T7噬菌体作为常被研究的模式生物, 其DNA聚合酶gp5在复制过程中既会参与链置换也会参与延伸合成, 已有的研究报道gp5自身独立链置换的能力很弱, 其与T7解旋酶gp4耦合形成复制体后可以发生快速且持续的链置换, 这一现象的分子机制尚待厘清. 本文通过单分子荧光共振能量转移(smFRET)的方法对gp5聚合过程的动力学进行了研究, 发现gp5在没有外力帮助下时会进入链置换-外切的循环, 导致聚合难以延伸, 调控这一循环的关键则是DNA双链退火压力. 进一步的实验表明gp5和gp4形成复制体后, gp4辅助gp5克服了退火压力从而聚合可以延伸.

     

    DNA polymerase is essential for DNA replication and repair. As it only performs the 5′-3′ polymerization, there are two kinds of DNA replication. One of them is called strand-displacement synthesis: DNA polymerase opens the double-strand (ds) DNA to attain the 3′-5′strand (leading strand) and copy this template in a continuous way, and the other is extension synthesis: DNA polymerase copies the newly separated 5′-3′ strand (lagging strand) in a discontinuous manner. The replication complex of T7 phage is an optimal model to investigate the mechanism of replication because it is only constituted by 4 terms of protein which are DNA helicase gp4, DNA polymerase gp5 with co-factor thioredoxin (Trx), and single-strand (ss) DNA-binding protein gp2.5. The replication complex of T7 encounters both strand-displacement synthesis and extension synthesis. Previous researches reported that gp5 can have rapid extension synthesis but lacks the ability to attain strand-displacement synthesis. It also reported that gp4 translocates on ssDNA at a rapid speed but unwinds dsDNA at a very low speed. However, gp5 and gp4 together can attain rapid and processive strand-displacement synthesis. Although extensively studied, this mechanism remains unclear. Here in this work, the dynamic of strand-displacement synthesis by gp5 is investigated with single-molecule Förster (fluorescence) resonance energy transfer (smFRET). It is found that gp5, without the help of external tension, can open dsDNA but only attain strand-displacement synthesis about 4 base pairs (bp), because its exonuclease activity excises the nascent nucleotides. Therefore gp5 repeats in the synthesis-excision cycle which results in the less production of strand-displacement synthesis. We conduct another control experiment by nano-tensioner, a high precision smFRET setup which can exert a tension on dsDNA, to change the dsDNA regression pressure on gp5. It is observed that reduced dsDNA regression pressure can increase the length of strand-displacement synthesis and reduce the length of excision which indicates that the dsDNA regression pressure can regulate the strand-displacement synthesis of gp5. The further experiment shows that after gp5 and gp4 are assembled into a replisome, it can have a processive strand-displacement synthesis and barely any excision presented. The speed of replisome is a little higher than gp5 alone but much higher than gp4 alone. Additionally, the length of strand-displacement synthesis by replisome is much longer than gp5 alone. Therefore it is indicated that the gp4 can reduce dsDNA regression pressure to enables gp5 to attain processive strand-displacement synthesis. On the other hand, the gp5 facilitates gp4 to unwind the dsDNA.

     

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