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

Jia Qi; Fan Qin-Kai; Hou Wen-Qing; Yang Chen-Guang; Wang Li-Bang; Wang Hao; Xu Chun-Hua; Li Ming; Lu Ying

doi:10.7498/aps.70.20210707

Abstract

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.

Keywords:

T7 DNA polymerase /
T7 DNA helicase /
fluorescence resonance energy transfer /
DNA regression pressure

Authors and contacts

1.
Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China
2.
University of Chinese Academy of Sciences, Beijing 100049, China

Corresponding author: Lu Ying, yinglu@iphy.ac.cn

Funds: Project supported by the National Science Foundation of China (Grant Nos. 12090051, 11834018, 12022409), the CAS Key Research Program of Frontier Sciences, China (Grant No. QYZDJ-SSW-SYS014), and the Youth Innovation Promotion Association of CAS (Grant No. 2017015)

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References

[1]	Benkovic S J, Valentine A M, Salinas F 2001 Annu. Rev. Biochem. 70 181 Google Scholar
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Cited By

图 1 exo⁺ gp5和exo^– gp5的PAGE实验　(a)电泳实验的DNA, 在引物链5′端标记有Alexa488; (b) 对Alexa488照明得到的结果: 第1个条带为DNA的原始长度, 第2个条带是exo⁺ gp5合成1 min后的结果, 第3个条带是exo^– gp5合成1 min后的结果, 第4个条带是exo⁺ gp5合成5 min后的结果, 第5个条带是exo^– gp5合成5 min后的结果

Figure 1. PAGE assays of exo⁺ gp5 and exo^– gp5. (a) Illustration of DNA that used in PAGE assays. Alexa488 is labeled on the 5′ overhand of primer DNA. (b) Results of various condition. The first lane: Original length of the DNA. The second lane: The synthesis-product of exo⁺ gp5 with 1 minute. The third lane: The synthesis-product of exo^– gp5 with 1 minute. The fourth lane: The synthesis-product of exo⁺ gp5 with 5 minute. The fifth lane: The synthesis-product of exo^– gp5 with 5 minute.

DownLoad: Full-Size Img PowerPoint

图 2 T7 DNA聚合酶gp5不断重复链置换和外切　(a), (b)没有结合辅助因子Trx时聚合酶无法链置换DNA双链; (c), (d)有辅助因子Trx时聚合酶能够部分链置换DNA, 但是会回退; (e), (f)外切活性突变后的gp5不会再外切; (g) exo⁺ gp5 + Trx实验中合成长度的统计图, 其分布满足单e指数; (h) exo⁺ gp5 + Trx实验中外切长度的统计图, 其分布满足单e指数

Figure 2. T7 DNA polymerase gp5 repeats in synthesis-excision cycle: (a), (b) exo⁺ gp5 cannot have displacement synthesis without co-factor Trx; (c), (d) gp5 with Trx repeats in synthesis-excision cycle; (e), (f) exo^– gp5 attain full-length displacement synthesis without excision; (g) histogram of synthesis processivity from assay of exo⁺ gp5 + Trx, the distribution is well fit by an exponential; (h) histogram of excision processivity from assay of exo⁺ gp5+ Trx, the distribution is well fit by an exponential.

DownLoad: Full-Size Img PowerPoint

图 4 T7 DNA解旋酶帮助聚合酶克服退火压力　(a) gp4, exo⁺ gp5和Trx共同链置换的示意图, (b) gp4, exo⁺ gp5和Trx共同链置换时的典型曲线; (c) exo⁺ gp5 + Trx + gp4可以完全链置换; (d)不同情况的链置换速度

Figure 4. gp4 decrease DNA regression pressure which facilitate gp5 to attain processive strand-displacement synthesis: (a) Illustration of displacement by gp4, exo⁺ gp5 and Trx; (b) typical trace from assay of gp4, exo⁺ gp5 and Trx; (c) exo⁺ gp5 + Trx + gp4 attain processive synthesis; (d) synthesis speed in various condition.

DownLoad: Full-Size Img PowerPoint

图 3 T7 DNA聚合酶gp5外切的原因是退火压力　(a)纳米张力器示意图; (b) 纳米张力器实验的典型曲线; (c) exo⁺ gp5 + Trx在受力后合成长度的统计图, 其分布满足单e指数; (d) exo⁺ gp5 + Trx受力后外切长度的统计图, 其分布满足单e指数

Figure 3. DNA regression pressure induced exonuclease activity: (a) Illustration of nanotensionior; (b) typical trace from assay of nanotensionior; (c) histogram of synthesis processivity from assay of exo⁺ gp5 + Trx with tension, the distribution is well fit by an exponential; (d) histogram of excision processivity from assay of exo⁺ gp5 + Trx with tension, the distribution is well fit by an exponential.

DownLoad: Full-Size Img PowerPoint

图 5 T7 DNA聚合酶不同情况链置换时的模型　(a) 野生型gp5单独链置换时进入链置换和外切的循环; (b) 野生型gp5在gp4的帮助下, 没有外切的出现可以持续链置换

Figure 5. Model for gp5 strand displacement activity in various condition: (a) exo⁺ gp5 repeats in synthesis-excision cycle; (b) gp4 facilitate exo⁺ gp5 to attain processive strand-displacement synthesis.

DownLoad: Full-Size Img PowerPoint

map

[1]	Benkovic S J, Valentine A M, Salinas F 2001 Annu. Rev. Biochem. 70 181 Google Scholar
[2]	O'Donnell M 2006 J. Biol. Chem. 281 10653 Google Scholar
[3]	Pandey M, Syed S, Donmez I, Patel G, Ha T, Patel S S 2009 Nature 462 940 Google Scholar
[4]	Sun B, Pandey M, Inman J T, Yang Y, Kashlev M, Patel S S, Wang M D 2015 Nat. Commun. 6 10260 Google Scholar
[5]	Kath J E, Jergic S, Heltzel J M, Jacob D T, Dixon N E, Sutton M D, Walker G C, Loparo J J 2014 Proc. Natl. Acad. Sci. U.S.A. 111 7647 Google Scholar
[6]	Nandakumar D, Pandey M, Patel S S 2015 Elife 4 e06562 Google Scholar
[7]	Pandey M, Patel S S 2014 Cell Rep. 6 1129 Google Scholar
[8]	Syed S, Pandey M, Patel S S, Ha T 2014 Cell Rep. 6 1037 Google Scholar
[9]	Gao Y, Cui Y, Fox T, Lin S, Wang H, de Val N, Zhou Z H, Yang W 2019 Science 363 eaav7003 Google Scholar
[10]	Stano N M, Jeong Y J, Donmez I, Tummalapalli P, Levin M K, Patel S S 2005 Nature 435 370 Google Scholar
[11]	Manosas M, Spiering M M, Ding F, Bensimon D, Allemand J F, Benkovic S J, Croquette V 2012 Nucleic Acids Res. 40 6174 Google Scholar
[12]	Lin W, Ma J, Nong D, Xu C, Zhang B, Li J, Jia Q, Dou S, Ye F, Xi X, Lu Y, Li M 2017 Phys. Rev. Lett. 119 138102 Google Scholar
[13]	Ma J B, Jia Q, Xu C H, Li J H, Huang X Y, Ma D F, Li M, Xi X G, Lu Y 2018 J. Phys. Chem. B 122 5790 Google Scholar
[14]	黄星榞, 隋明宇, 侯文清, 李明, 陆颖, 徐春华 2020 69 208706 Google Scholar Huang X Y, Sui M Y, Hou W Q, Li M, Lu Y, Xu C H 2020 Acta Phys. Sin. 69 208706 Google Scholar
[15]	Kim D E, Narayan M, Patel S S 2002 J. Mol. Biol. 321 807 Google Scholar
[16]	Ma J B, Chen Z, Xu C H, Huang X Y, Jia Q, Zou Z Y, Mi C Y, Ma D F, Lu Y, Zhang H D, Li M 2020 Nucleic Acids Res. 48 3156 Google Scholar
[17]	Schwartz J J, Quake S R 2009 Proc. Natl. Acad. Sci. U.S.A. 106 20294 Google Scholar
[18]	Ha T 2001 Methods 25 78 Google Scholar
[19]	Etson C M, Hamdan S M, Richardson C C, van Oijen A M 2010 Proc. Natl. Acad. Sci. U.S.A. 107 1900 Google Scholar
[20]	Ibarra B, Chemla Y R, Plyasunov S, Smith S B, Lazaro J M, Salas M, Bustamante C 2009 EMBO J. 28 2794 Google Scholar
[21]	Hoekstra T P, Depken M, Lin S N, Cabanas-Danes J, Gross P, Dame R T, Peterman E J G, Wuite G J L 2017 Biophys. J. 112 575 Google Scholar
[22]	Johansson E, Dixon N 2013 Cold Spring Harb. Perspect. Biol. 5 a012799 Google Scholar
[23]	Derbyshire V, Freemont P S, Sanderson M R, Beese L, Friedman J M, Joyce C M, Steitz T A 1988 Science 240 199 Google Scholar
[24]	Lam W C, Van der Schans E J, Joyce C M, Millar D P 1998 Biochemistry 37 1513 Google Scholar
[25]	Graham J E, Marians K J, Kowalczykowski S C 2017 Cell 169 1201 Google Scholar
[26]	陈泽, 马建兵, 黄星榞, 贾棋, 徐春华, 张慧东, 陆颖 2018 67 118201 Google Scholar Chen Z, Ma J-B, Huang X Y, Jia Q, Xu C H, Zhang H D, Lu Y 2018 Acta Phys. Sin. 67 118201 Google Scholar

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