A Light‐Activated DNA Polymerase

Chou, Chin Cheng; Young, Douglas D.; Deiters, Alexander

doi:10.1002/ange.200901115

Cited by 30 publications

(26 citation statements)

References 24 publications

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“…[20] A further possibility is a light-activated polymerase, previously described for the bacterial polymerase from Thermus aquaticus, by using in vivo incorporation of a photolabile tyrosine analogue. [21] Such derivatives, which are more easily prepared by modification of a suitable cysteine with a photolabile group, are useful for studying polymerase mechanisms and in hot-start PCR.…”

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confidence: 99%

Role of Disulfide Bridges in Archaeal Family‐B DNA Polymerases

Killelea

Connolly

2011

ChemBioChem

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The family-B DNA polymerases obtained from the order Thermococcales, for example, Pyrococcus furiosus (Pfu-Pol) are commonly used in the polymerase chain reaction (PCR) because of their high thermostability and low error rates. Most of these polymerases contain four cysteines, arranged as two disulfide bridges. With Pfu-Pol C429-C443 forms one of the disulfides (DB1) and C507-C510 (DB2) the other. Although the disulfides are well conserved in the enzymes from the hyperthermophilic Thermococcales, they are less prevalent in euryarchaeal polymerases from other orders, and tend to be only found in other hyperthermophiles. Here, we report on the effects of deleting the disulfide bridges by mutating the relevant cysteines to serines. A variety of techniques, including differential scanning calorimetry and differential scanning fluorimetry, have shown that both disulfides make a contribution to thermostability, with DB1 being more important than DB2. However, even when both disulfides are removed, sufficient thermostability remains for normal (identical to the wild type) performance in PCR and quantitative (real-time) PCR. Therefore, polymerases totally lacking cysteine are fully compatible with most PCR-based applications. This observation opens the way to further engineering of polymerases by introduction of a single cysteine followed by appropriate chemical modification.

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confidence: 99%

Role of Disulfide Bridges in Archaeal Family‐B DNA Polymerases

Killelea

Connolly

2011

ChemBioChem

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“…[12, 17] We have developed a light-activated bacteriophage T7 RNA polymerase through the introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site. The caged polymerase enzyme is expressed in bacterial cells engineered with an additional cognate tRNA/tRNA synthetase; this enables the production of substantial quantities of this protein.…”

Section: Discussionmentioning

confidence: 99%

“…[12] Irradiation of the caged Taq DNA polymerase with UV light of 365 nm for 5 minutes restored 67 % of activity compared to the wild-type enzyme. Accordingly, we hypothesized that introduction of the caged tyrosine 1 , bearing an ONB group, at the Tyr639 position of T7 RNA polymerase would inactivate it by occupying the space reserved for the incoming NTP with the sterically demanding ONB group (Figure 2C and D).…”

Section: Introductionmentioning

confidence: 99%

Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro‐ and Eukaryotic Cells

2010

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A light-activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site-specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradiation with nonphototoxic UV light of 365 nm removes the ortho-nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro- and eukaryotic systems allowed for the photochemical activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradiation of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradiation with UV light led to the activation of T7RNAP, thereby inducing RNA polymerization and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.

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“…Optochemical control studies began with the incorporation of 21 into b-galactosidase and 2-nitrobenzylcysteine (31) into caspase-3, respectively, to monitor their enzymatic activities [62,63]. Likewise, incorporation of 21 into Taq DNA polymerase successfully enabled researchers to regulate the enzyme activity [64]. Later, 2-nitrobenzyllysine (32) was introduced at the active site of firefly luciferase to facilitate the measurement of intracellular ATP dynamics in HEK293T cells [65].…”

Section: Photo-controllable Enzymesmentioning

confidence: 99%