Strand displacement DNA synthesis by DNA polymerase gp90 exo― of Pseudomonas aeruginosa phage 1

Mi, Chenyang; Zhang, Shuming; Huang, Weihuan; Dai, Mengyuan; Chai, Zili; Wang, Yang; Deng, Shanshan; Ao, Lin; Zhang, Huidong

doi:10.1016/j.biochi.2019.12.013

Cited by 3 publications

(10 citation statements)

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“…Nucleic acids' ability to form hydrogen bonds between complementary Watson-Crick bases allows for a rich set of complicated, multi-step kinetic behaviors such as duplex hybridization [1] and dehybridization [2], Holliday junction structural dynamics [3,4], and strand invasion [5]. In particular, strand displacement, which is the exchange of bases between two competing nucleic acid strands of identical sequence, occurs in homologous recombination [6,7], DNA replication [8] and RNA transcription [9], as well as CRISPR/Cas [10] and the related Cascade complex [11]. In addition to fundamental genomic processes, DNA nanotechnology exploits strand displacement to create nanoscale gadgets [12][13][14] and computational circuits [15][16][17][18].…”

Section: Introductionmentioning

confidence: 99%

First passage time study of DNA strand displacement

Broadwater

Cook

Kim

2020

Preprint

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6DNA strand displacement, where a single-stranded nucleic acid invades a DNA duplex, is perva-7 sive in genomic processes and DNA engineering applications. The kinetics of strand displacement 8 have been studied in bulk; however, the kinetics of the underlying strand exchange were obfus-9 cated by a slow bimolecular association step. Here, we use a novel single-molecule Fluorescence 10 Resonance Energy Transfer (smFRET) approach termed the "fission" assay to obtain the full dis-11 tribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, 12 the first passage time distribution for a 14-nt displacement domain exhibited a nearly monotonic 13 decay with little delay. Among the eight different sequences we tested, the mean displacement time 14 was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also 15 varied between complementary invaders and between RNA and DNA invaders of the same base 16 sequence except for T→U substitution. However, displacement times were largely insensitive to 17 the monovalent salt concentration in the range of 0.25 M to 1 M. Using a one-dimensional random 18 walk model, we infer that the single-step displacement time is in the range of ∼30 µs to ∼300 µs 19 depending on the base identity. The framework presented here is broadly applicable to the kinetic 20 analysis of multistep processes investigated at the single-molecule level. 21 * These two authors contributed equally. † harold.kim@physics.gatech.edu 1 Nucleic acids' ability to form hydrogen bonds between complementary Watson-Crick 2 bases allows for a rich set of complicated, multi-step kinetic behaviors such as duplex 3 hybridization[1] and dehybridization[2], Holliday junction structural dynamics[3, 4], and 4 strand invasion[5]. In particular, strand displacement, which is the exchange of bases 5 between two competing nucleic acid strands of identical sequence, occurs in homologous 6 recombination[6, 7], DNA replication[8] and RNA transcription[9], as well as CRISPR/Cas[10] 7 and the related Cascade complex[11]. In addition to fundamental genomic processes, DNA 8 nanotechnology exploits strand displacement to create nanoscale gadgets[12-14] and com-9 putational circuits[15-18]. Strand displacement also aids in the development of quantitative 10 assays for detection of nucleic acid[19-21] and enzymatic activity[22, 23] with improved 11 probe specificity[24-26]. 12For practical applications, strand displacement is implemented with the "invader" 13 strand and a partial duplex composed of the "incumbent" strand and the "substrate" 14 strand[18] (Fig. 1). The partial duplex has two distinct domains: 1) the single-stranded 15 overhang called the toehold which is critical to the speed and efficiency of the reaction[27] 16 and 2) the duplex region called the displacement domain. Toehold-mediated strand dis-17 placement is initiated when the invader strand anneals to the toehold in a bimolecular 18 reaction. Once a stable toehold interaction is formed, the incumben...

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Section: Introductionmentioning

confidence: 99%

First passage time study of DNA strand displacement

Broadwater

Cook

Kim

2020

Preprint

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“…37 It has been shown that gp90 exobinds to DNA fork containing a flap and a gap with high affinity. 12 Significantly, in the presence of gp90 exo -(Figure 1C), the unzipping force increases to ~17 pN (Figure 1D). The distribution of unzipping force is centered at 17.1 ± 2.4 pN (mean ± SD, N = 155) (Figure 1E).…”

Section: Dna Hairpinmentioning

confidence: 95%

“…8,9 In general, SDDS shows a lower processivity and rate compared with primer extension (PE). 10,11 Previous studies have revealed that high GC content 12,13 and protein binding 14,15 in the downstream duplex inhibits SDDS. Recently, our group has identified that DNA polymerase gp90 from Pseudomonas aeruginosa phage 1 (PaP1) is an A-family processive DNA polymerase containing 3′-5′ Exo activities.…”

Section: Introductionmentioning

confidence: 99%

“…16 Both wild-type (wt) and exonuclease-deficient gp90 (gp90 exo -) perform SDDS efficiently (Figure S1A). 12 As the bulky assays revealed, a tail (≥20 nt) and a gap (1-5 nt) at DNA fork is essential for SDDS. 12 However, the detailed effects of exonuclease activity, tension, GC content, and dNTP concentration on SDDS are still elusive.…”

Section: Introductionmentioning

confidence: 99%

“…12 As the bulky assays revealed, a tail (≥20 nt) and a gap (1-5 nt) at DNA fork is essential for SDDS. 12 However, the detailed effects of exonuclease activity, tension, GC content, and dNTP concentration on SDDS are still elusive. The answers to these critical questions will give a deeper understanding of SDDS mechanism.…”

Section: Introductionmentioning

confidence: 99%

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DNA polymerase Gp90 activities and regulations on strand displacement DNA synthesis revealed at single‐molecule level

Zhang

Xiao

et al. 2021

FASEB j.

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Strand displacement DNA synthesis (SDDS) is an essential step in DNA replication.With magnetic tweezers, we investigated SDDS kinetics of wild-type gp90 and its exonuclease-deficient polymerase gp90 exoat single-molecule level. A novel binding state of gp90 to the fork flap was confirmed prior to SDDS, suggesting an intermediate in the initiation of SDDS. The rate and processivity of SDDS by gp90 exoor wt-gp90 are increased with force and dNTP concentration. The rate and processivity of exonuclease by wt-gp90 are decreased with force. High GC content decreases SDDS and exonuclease processivity but increases exonuclease rate for wt-gp90. The high force and dNTP concentration and low GC content facilitate the successive SDDS but retard the successive exonuclease for wt-gp90. Furthermore, increasing GC content accelerates the transition from SDDS or exonuclease to exonuclease. This work reveals the kinetics of SDDS in detail and offers a broader cognition on the regulation of various factors on SDDS at single-polymerase level.

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