KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry

Betz, Karin; Malyshev, Denis A.; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Dwyer, Tammy J.; Ordoukhanian, Phillip; Romesberg, Floyd E.; Marx, Andreas

doi:10.1038/nchembio.966

Cited by 143 publications

(196 citation statements)

References 25 publications

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“…1A) being a particularly promising example (5-7). Despite lacking complementary hydrogen bonding, we demonstrated that the dNaM-d5SICS UBP is well replicated by a variety of DNA polymerases in vitro (7-10), and that this efficient replication is mediated by a unique mechanism that draws upon interbase hydrophobic and packing interactions (11,12). These efforts then culminated in the first progress toward the creation of an SSO in 2014, when we reported that Escherichia coli grown in the presence of the corresponding unnatural nucleoside triphosphates (dNaMTP and d5SICSTP), and provided with a plasmid-encoded nucleoside triphosphate transporter (NTT2) from Phaeodactylum tricornutum (which we denote as PtNTT2) (13), is able to import the unnatural triphosphates and replicate a single dNaM-d5SICS UBP on a second plasmid (14).…”

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confidence: 99%

A semisynthetic organism engineered for the stable expansion of the genetic alphabet

Zhang

Lamb

Feldman

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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All natural organisms store genetic information in a four-letter, twobase-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chemically optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.T he natural genetic alphabet is composed of four letters whose selective pairing to form two base pairs underlies the storage and retrieval of virtually all biological information. This alphabet is essentially conserved throughout nature, and has been since the last common ancestor of all life on Earth. Significant effort has been directed toward the development of an unnatural base pair (UBP), formed between two synthetic nucleotides, that functions alongside its natural counterparts (1-3), which would represent a remarkable integration of a man-made, synthetic component into one of life's most central processes. Moreover, semisynthetic organisms (SSOs) that stably harbor such a UBP in their DNA could store and potentially retrieve the increased information, and thereby lay the foundation for achieving the central goal of synthetic biology: the creation of new life forms and functions (4).For over 15 years, we have sought to develop such a UBP (1), and these efforts eventually yielded a family of predominantly hydrophobic UBPs, with that formed between dNaM and d5SICS (dNaM-d5SICS; Fig. 1A) being a particularly promising example (5-7). Despite lacking complementary hydrogen bonding, we demonstrated that the dNaM-d5SICS UBP is well replicated by a variety of DNA polymerases in vitro (7-10), and that this efficient replication is mediated by a unique mechanism that draws upon interbase hydrophobic and packing interactions (11,12). These efforts then culminated in the first pro...

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confidence: 99%

A semisynthetic organism engineered for the stable expansion of the genetic alphabet

Zhang

Lamb

Feldman

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

155

208

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“…Similarly, in a NMR study on 12-mer 5'-CCTTTCD5SICSTTCTC-3' reveals solution-conformation of the possibility [9]. The structural conformational details of the study have reached on following conclusions [8,10] (i) The mechanism of the insertion of DNAMTP opposite d5SICS was less efficient than the insertion of D5SICSTP opposite DNAM in starting replication. (ii) The nucleobases paired in an intercalated manner, similar to their pairing in free duplex DNA and the mode of pairing depends on the length of the single-stranded template, and (iii) more importantly, the mode of intercalation appeared to depend mostly on sequencespecific interactions of the flanking nucleotides, with the specific packing interactions between the intercalating nucleobases of secondary importance.…”

Section: Fig 1: Dsics-mmo2 and D5sics-dnam Base-pairmentioning

confidence: 99%

“…Romesberg et al [7,8] has successfully engineered replications of some sequences of nucleic acid using hydrophobic base pairs, and they found 60 base-pairs out of about 3,600 combinations which prove the earlier principle to some extent. The base-pair DMMO2-DSICS was found more appropriate for pairing as it shows two contradictory characteristics, hydrophobic for enzymes into DNAfunction and accepts hydrogen-bond when enzymes are replicating the strand.…”

Section: Fig 1: Dsics-mmo2 and D5sics-dnam Base-pairmentioning

confidence: 99%

“…In the refining process of these base-pairs the base-pair DNAM-D5SICS ( fig. 1) was found to be most efficient which was synthesized by modifying base-pair DMMO2-DSICS [8] Its hydrophobic characteristics maintained helix integrity and hydrophilic the central dogma function; just like switching device at the key position of the mechanism of the central-dogma [7] The profound insight, received about modified base-pair DNAM-D5SICS, has been utilized in the study of some crystal structures of KlenTaq-DNA-polymerase which was the large fragment of the type I DNA-polymerase from ThermusAquaticus when made the complex with a templating DNAM with or without bound D5SICSTP [9]. Similarly, in a NMR study on 12-mer 5'-CCTTTCD5SICSTTCTC-3' reveals solution-conformation of the possibility [9].…”

Section: Fig 1: Dsics-mmo2 and D5sics-dnam Base-pairmentioning

confidence: 99%

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MOLECULAR DYNAMICS STUDY OF NON-HYDROGEN-BONDING BASE-PAIR DNA DUPLEX d(GTCDNAM GCGCCGTGGC). d(GCCACGGCGCD5SICSGAC)

Tiwari

2018

Int J Pharm Pharm Sci

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Objective: The objective of the study was to elucidate the structural activity of a natural DNA sequence modified by a hydrophobic base-pair which didn't form Watson-Crick (W-C) hydrogen-bond. The modified unnatural base-pair (DNAM-D5SICS) was introduced in DNA sequences of 14-mer for molecular dynamics study in water solution. Methods:A 200 ns molecular-dynamics-simulation in orthogonal-box-water-solvent, using Particle-mesh-Ewald-method (PME) within periodicboundary-condition (PBC) was performed by using AMBER-14 code. The force-field-ff12SB-force-field was used during the simulation while the force-field-parameters of modified base-pair, compatible to ff12SB-force-field, were calculated by Gaussian-09-code using ab-inito/Hartree-Fockmethodology. The code CPPTRAJ, (a module of AMBER-14) CURVE and Chimera were used in the analysis of the data.Results: Root mean square deviation (RMSD) of heavy atoms of the trajectory revealed that the structure of the equilibrated duplex was stable, sequence-dependent and had mixed DNA-conformation. A little distortion near to the neighbor of the modified base-pair in the duplex strand was observed. However, we got a stabilized structure of such type of duplex if we placed modified base-pair after the third place in the strand. Conclusion:The study concluded that the distortion produced by modified-base-pair in the overall structure of duplex was local while the confirmation of such type of duplex was mixed and maintained the Watson-Crick (W-C) integrity of DNA. The study would help in the use of hydrophobic base-pair materials in biotechnological applications and the understanding of their structure-function relationship.

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“…Recent structural and functional data employing hydrophobic nucleobase surrogates with increased stacking ability reveal that DNA polymerase active sites are not only capable of selecting for a correct structure among the pairing nucleotides but also, at least in some cases, capable of enforcing it. [33] This suggests that DNA polymerases might have evolved to favor a coplanar geometry to prevent natural nucleotide mispairing via cross-strand intercalation and instead allow only the more specific, edge-to-edge hydrogen-bonding interactions of the Watson-Crick type. [33] …”

Section: Minor Groove Scanningmentioning

confidence: 99%

Getting it Right: How DNA Polymerases Select the Right Nucleotide

Ludmann

Marx²

2016

Chimia

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All living organisms are defined by their genetic code encrypted in their DNA. DNA polymerases are the enzymes that are responsible for all DNA syntheses occurring in nature. For DNA replication, repair and recombination these enzymes have to read the parental DNA and recognize the complementary nucleotide out of a pool of four structurally similar deoxynucleotide triphosphates (dNTPs) for a given template. The selection of the nucleotide is in accordance with the Watson-Crick rule. In this process the accuracy of DNA synthesis is crucial for the maintenance of the genome stability. However, to spur evolution a certain degree of freedom must be allowed. This brief review highlights the mechanistic basis for selecting the right nucleotide by DNA polymerases.

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KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry

Cited by 143 publications

References 25 publications

A semisynthetic organism engineered for the stable expansion of the genetic alphabet

A semisynthetic organism engineered for the stable expansion of the genetic alphabet

MOLECULAR DYNAMICS STUDY OF NON-HYDROGEN-BONDING BASE-PAIR DNA DUPLEX d(GTCDNAM GCGCCGTGGC). d(GCCACGGCGCD5SICSGAC)

Getting it Right: How DNA Polymerases Select the Right Nucleotide

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