Polymerase Interactions with Wobble Mismatches in Synthetic Genetic

Subscriber access provided by University of Sussex Library

Rapid Report

Polymerase Interactions with Wobble Mismatches in Synthetic Genetic Systems and their Evolutionary Implications Christian B. Winiger, Myong-Jung Kim, Shuichi Hoshika, Ryan W Shaw, Jennifer D. Moses, Mariko F. F. Matsuura, Dietlind L. Gerloff, and Steven A. Benner Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00533 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on July 7, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Polymerase Interactions with Wobble Mismatches in Synthetic Genetic Systems and their Evolutionary Implications Christian B. Winiger a, Myong-Jung Kima,b, Shuichi Hoshikaa,b, Ryan W. Shawa,b, Jennifer D. Mosesa,b, Mariko F. Matsuuraa,c, Dietlind L. Gerloffa, and Steven A. Bennera,b* a

Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Blvd. Box 7, Alachua, FL 32615, USA

b

Firebird Biomolecular Sciences LLC, 13709 Progress Blvd. Box 17, Alachua, FL 32615, USA

c

Department of Chemistry, University of Florida, Gainesville, FL 32611, USA

KEYWORDS: artificially expanded genetic information systems, wobble base pairs, evolution, polymerase, synthetic biology, DNA Supporting Information Placeholder ABSTRACT: In addition to completing the Watson-Crick nucleobase matching “concept” (big pairs with small, hydrogen bond donors pair with hydrogen bond acceptors), artificially expanded genetic information systems (AEGIS) also challenge DNA polymerase 1 from E. coli with a complete set of mismatches, including wobble mismismatches. Here, we explore wobble mismatches with AEGIS. Remarkably, we find that the polymerase tolerates a AEGIS:standard wobble that has the same geometry as the G:T wobble that polymerases have evolved to exclude, but excludes a wobble geometry that polymerases have never encountered in natural history. These results suggest certain limits to “structural analogy” and “evolutionary guidance” as tools to help synthetic biologists expand DNA alphabets.

mispairing. In addition to its scientific interest, this understanding is important for efforts to apply AEGIS in biomedical technology and to move AEGIS into living cells. a)

X

K

2-aminoadenine:T

G:T Type 1 wobble

b)

X:T Type 1 wobble

X:4-ThioT Type 1 wobble

X:2-ThioT Type 1 wobble

A:isoC Me Type 1 wobble

X:T Type 2 wobble

X:4-ThioT Type 2 wobble

X:2-ThioT Type 2 wobble

2-aminopurine:C Type 2 wobble

c)

Synthetic biologists seek to construct artificial systems that reproduce behaviors valued in life, such as replication, adaptation, and evolution, but with molecules different from those found in nature. Following Feynman’s dictum (“What I cannot make, I do not understand”), this approach allows the biochemist to better understand the intimate connection between molecular behavior and biology, and distinguish between understanding and the illusion of understanding. Artificially expanded genetic information systems (AEGIS) are examples of unnatural molecules that have come from this effort. AEGIS contains additional, independently replicable nucleobases that pair via a natural Watson-Crick geometry but with unnatural hydrogen bonding patterns.1-6 By shifting the hydrogen donor and acceptor positions of nucleobases, a total of 12 potential AEGIS nucleobases is possible, adding an extra four nucleobase pairs to the genetic alphabet.7 AEGIS in various forms has been shown to be replicable, evolvable, and adaptable, at least in vitro.8-13 However, in addition to completing the Watson-Crick pairing “concept”, AEGIS also completes the range of possible hydrogen bonded mispairs. This allows AEGIS to serve as a platform to better understand how natural systems handle

Figure 1. a) Watson-Crick pairs between the xanthosine analogue X and K and 2,6-diaminoadenine and T. The only wobble base pair that is possible with the standard nucleobase set is that between G and T with a Type 1 wobble geometry. b).Type 1 wobbles between X and T (or its analogs) and Type 1 wobble between A and isoCMe c) Type 2 wobbles between X and T (or its analogs) and the Type 2 wobble between 2-aminopurine and C.

Here, we explore mismatching with an analogue of xanthosine 8-(β−D-2'-deoxyribofuranosyl) imidazo[1,2-a]-1,3,5triazine-2(8H)-4(3H)-dione (trivially X) and its Watson-Crick partner 5-(β−D-2'-deoxyribo-furanosyl)-2,4-diamino pyrimidine (trivially K, Figure 1a),14,15 examining how these are managed by DNA Polymerase 1 from E. coli, a Family A polymerase. The K:X pair is “symmetrical”, in that it is joined by three hydrogen bonds with the big component (X) having an “acceptor-donor-acceptor” (ADA) pattern. This is the same “symmetry” displayed by standard T, a “symmetry” that is broken in the standard T:A pair because A lacks a 2-position amino group.

ACS Paragon Plus Environment

Biochemistry Templates containing X (Figure 2a) were synthesized and purified by PAGE. In parallel, the triphosphate of dK (dKTP), the AEGIS Watson-Crick complement of dX, was synthesized using published procedures.16 DNA polymerase I carrying a His tag was purified from E. coli culture by cobalt-affinity chromatography. Polymerase concentrations were determined using a Bradford assay.17

a)

annealed template/primer/probe

3‘ BH2/ GAA AGC GAG CGA CGG AGC CAG GA CCG CCA CCT CCG CC 5‘ JOE/ CTT TCG CTC GCT GCC TCG GTC CTG TTG TTX GGC GGT GGA GGC GGT

b) 1050

fluorescence [a.u.]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

850

650

450

250

50 0

1000

2000

3000

4000

5000

Page 2 of 5

As both X and T present ADA hydrogen bonding patterns, it seemed unlikely that the X:T pair adopted a standard Watson-Crick geometry (three clashes). However, the symmetry of both nucleobases allows them to form two wobble pairs (Figure 1 b-c), Type 1 (pyrimidine displaced towards the major groove) and Type 2 (pyrimidine displaced towards the minor groove. To determine which wobble is allowed, we exploited the well-studied weakness of C=S groups to serve as hydrogen bond acceptors, when compared to C=O groups.19-28 The sulfur of 4-ThioTTP should disfavor the Type 2 wobble while allowing a Type 1 wobble (Figure 1b). Conversely, 2-ThioTTP should disfavor the Type 1 wobble while allowing a Type 2 wobble (Figure 1c). Therefore, these thio analogs can probe which wobble might allow misincorporation of T opposite template dX. In fact, when the polymerase was challenged with 4ThioTTP, signal was formed at the same rate as when template X was complemented by TTP (Figure 2b). However, when polymerase was challenged to mismatch 2-ThioTTP opposite dX, primer extension failed. From this, we inferred that this polymerase tolerates a Type 1 wobble, but not a Type 2 wobble (Figure 1 b-c). Separately, the polymerase is known to accept 2-ThioTTP as a partner for template A (see Figure SI 1).20

time [s] no enzyme no dNTPs dATP, dGTP, dCTP dATP, dGTP, dCTP, dTTP dATP, dGTP, dCTP, dKTP dATP, dGTP, dCTP, 2-thioTTP dATP, dGTP, dCTP, 4-thioTTP

Figure 2. a) Sequence of the annealed template, primer and probe used in the 5’ nuclease assay. b) Incorporation of a nucleotide opposite of X and extension of the primer by polymerase I (in 5’ → 3’ direction) leads to digestion of the annealed probe (bearing quencher) and increasing fluorescence. dNTPs was 0.1 mM in each reaction are indicated.

To measure matching and mismatching in the GACTKX system, a TaqMan-like assay was designed to exploit the 5’nuclease activity of polymerase I18, a template tagged with 5’dichlorodimethoxyfluorescein, a primer, and a probe tagged by 3’ BlackHole-Quencher® II. The nucleotide to be probed (X) was placed only once in the template. The template also lacked dA, to facilitate mismatch analysis. Incorporation of a nucleotide opposite X allows strand synthesis to be completed. This, in turn, allows the nuclease to digest the probe, separating quencher from fluor to create a signal (Figure 2). As expected, generation of a full fluorescent signal is seen when dKTP is added to a mixture containing primer, template, and probe in the presence of the polymerase, dCTP, dGTP, and dATP (Figure 2b). This shows that the polymerase can synthesize duplexes with the K:X AEGIS Watson-Crick match. However, when dKTP was omitted but dTTP was present, primer extension occurred in the assay at nearly the same rate (Figure 2b). Absent both dKTP and dTTP, primer extension failed. These data implied that this polymerase mismatches dTTP opposite template X during primer extension if dKTP is absent, but no other nucleotide.

Figure 3. Primer extension with 5’ labelled primer annealed to template (indicated in line P). Each reaction contained 0.2 mM of each indicated dNTP: (1) dATP, dGTP, dCTP (2) dATP, dGTP, dCTP, dTTP (3) dATP, dGTP, dCTP, dKTP (4) dATP, dGTP, dCTP, 2-ThioTTP (5) dATP, dGTP, dCTP, 4-ThioTTP. The 10 bp and 40 bp bands of the 10 bp ladder are marked (left).

Standing start extensions confirmed this (Figure 3). When the radiolabeled primer was extended in mixtures containing dATP, dCTP, dGTP and either dKTP, dTTP or 4-ThioTTP, full-length product was formed. Indeed, full-length product was formed faster with 4-ThioTTP than with dTTP. However, with 2-ThioTTP, no extension product was seen in the gel,

ACS Paragon Plus Environment

Page 3 of 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

and the same result was seen in experiments lacking both dKTP and dTTP (Table SI1). Polymerases can, of course, prevent mismatches in the elongation step and, later, by proofreading. The overall mismatching examined here, most relevant for synthetic biology goals, examines these two processes together. We also examined mismatching with the Klenow fragment of Pol 1, which lacks a proofreading 3’5’ exonuclease domain. Klenow made numerous “mistakes”, putting in A, G, and C opposite X (Figure 3), following by stalling. This suggests that the toleration of wobbles by the full DNA polymerase 1 reflects a failure of proofreading to remove X:T or X:4-ThioT mismatches that were introduced through mismatching in the primer extension step. From a synthetic biology perspective, these results suggests that successful incorporation of K:X pair into living E. coli cells will require ensuring that dKTP is always present in sufficient amount to ensure that the correct K:X pair is made. As an alternative, we might engineer the polymerase to change its ability to exclude X:T mismatches.29 As another alternative, T might be replaced by 2ThioT in the entire genetic system; analogous replacements are now being frequently contemplated.22,30,31 However, these results have implications for the evolutionary logic needed to guide such “grand challenge” research in synthetic biology. Molecular evolution suggests that the substrate specificity of enzymes can directly evolve to exclude only those molecules physiologically present during natural history. For example aminoacyl tRNA synthetases rigorously exclude incorrect amino acids present naturally, but are agnostic with respect to unnatural amino acids.32,33 Unnatural amino acids may nevertheless be excluded, but only to the extent that they are “structurally analogous” to an excluded natural amino acid. However, “structural analogy” is an imprecisely defined concept that reflects more the perspective of the analogizer than the analogized.

The structural basis for these patterns of tolerance and rejection is not clear. Inspection of crystal structures suggests the possibility of contacts from the polymerase to the DNA in the minor groove, possibly mediated by minor groove water,. These might constrain wobble geometries (see SI Figure 2), but alternative explanations are possible. In this context, it is interesting to note reports of viruses that use 2-aminoadenine instead of adenine in their genetic molecule (Figure 1a),37 and this report has been confirmed in the Marlière laboratory38,39. 2-Aminoadenine, like 2aminopurine, can form a Type 2 wobble with C. Thus, polymerases from these viruses do have an evolutionary reason to exclude Type 2 wobbles. Of course, it is interesting to speculate that adenine (instead of 2-aminoadenine) is used in most DNA because adenine (unlike 2-aminoadenine) cannot form a wobble with C. If this speculation is accepted, it would suggest that 2aminoadenine (not adenine) actually was the ancestral genetic component, and the DNA of these cyanophages has the primitive ancestral structure. In this case, the ability of both Family A and Family B to exclude Type 2 wobbles might be regarded as a vestige of their emerging at a time where genetic information was stored in a different molecular system. In our, view, however, the stronger inference is that we do not clearly understand “structural analogy” in an evolutionary context. This is also an important cautionary conclusion.

ASSOCIATED CONTENT Supporting Information. Materials and methods. Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author

Following this rationale, we do expect polymerases to have evolved to exclude unnatural Type 1 wobbles, because they must have evolved to exclude natural Type 1 wobbles (the G:T wobble).34 Conversely, we do not expect polymerases to have evolved to exclude unnatural Type 2 wobbles, because they never have seen a Type 2 wobble in their natural history.

Steven A. Benner [email protected] Tel: +1 386 418 8085; Fax: +1 844 259 6519

Unfortunately (for this logic), with the X:T system, we find the exact opposite. This polymerase excludes the Type 2 wobble that it has had no evolutionary reason to exclude, but tolerates the Type 1 wobble that it has had every reason to have evolved to exclude.

Author Contributions

This is not the first time that a similar phenomenon has been seen, although its implications on the value of evolutionary logic do not appear to have been previously noted. Two different publications separately report a Type 1 wobble between adenine and 5-methyl-isocytosine (another AEGIS component) and a Type 2 wobble between 2-aminopurine and cytosine (see Figure 1). Here, native polymerases from evolutionary Family B (RB69, T4 and Pfu) reject the “unnatural” 2-aminopurine:C wobble (Type 2),35 but tolerate the “natural” A:isoCMe wobble (Type 1).36 Thus, this Family B polymerase rejects the Type 2 wobble analogous to the Type 2 X:T wobble that our Family A polymerase rejects, also with no evident evolutionary reason.

Present Addresses Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Blvd. Box 17, Alachua, FL 32615, USA CBW did the experiments with the polymerase and led the team in their interpretation, MJK synthesized the AEGIS dKTP and AEGIS phosphoramidites, SH made the AEGIS oligonucleotides, RWS and JDM prepared and characterized the polymerase, DLG did the modeling, and SAB helped prepare the manuscript. All authors assisted in the design of the experiments.

Funding Sources We are indebted the US National Science Foundation (MCB1412869) and The Templeton World Charity Foundation for support of this work. We are also grateful to support from the Nucleic Acids Licensing and Firebird Biomolecular Sciences LLC. CBW acknowledges a grant from the Swiss National Science Foundation. Grant No. P2BEP3_158965

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

REFERENCES (1) Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A. Nature 1990, 343, 33-37. (2) Switzer, C.; Moroney, S. E.; Benner, S. A. J. Am. Chem. Soc. 1989, 111, 8322-8323. (3) Malyshev, D. A.; Romesberg, F. E. Angew. Chem.Int. Edit. 2015, 54, 11930-11944. (4) Yang, Z. Y.; Sismour, A. M.; Sheng, P. P.; Puskar, N. L.; Benner, S. A. Nucleic Acids Res. 2007, 35, 4238-4249. (5) Yang, Z. Y.; Hutter, D.; Sheng, P. P.; Sismour, A. M.; Benner, S. A. Nucleic Acids Res. 2006, 34, 6095-6101. (6) Benner, S. A. Accounts Chem. Res. 2004, 37, 784797. (7) Benner, S. A.; Sismour, A. M. Nat. Rev. Genet. 2005, 6, 533-543. (8) Sefah, K.; Yang, Z.; Bradley, K. M.; Hoshika, S.; Jimenez, E.; Zhang, L.; Zhu, G.; Shanker, S.; Yu, F.; Turek, D.; Tan, W.; Benner, S. A. Proceedings of the National Academy of Sciences of the United States of America 2014, 111, 14491454. (9) Hernandez, A. R.; Shao, Y.; Hoshika, S.; Yang, Z.; Shelke, S. A.; Herrou, J.; Kim, H.-J.; Kim, M.-J.; Piccirilli, J. A.; Benner, S. A. Angew. Chem.-Int. Edit. 2015, 54, 9853-9856. (10) Georgiadis, M. M.; Singh, I.; Kellett, W. F.; Hoshika, S.; Benner, S. A.; Richards, N. G. J. J. Am. Chem. Soc. 2015, 137, 6947-6955. (11) Leal, N. A.; Kim, H.-J.; Hoshika, S.; Kim, M.-J.; Carrigan, M. A.; Benner, S. A. ACS Synthetic Biology 2015, 4, 407-413. (12) Zhang, L.; Yang, Z.; Sefah, K.; Bradley, K. M.; Hoshika, S.; Kim, M.-J.; Kim, H.-J.; Zhu, G.; Jimenez, E.; Cansiz, S.; Teng, I. T.; Champanhac, C.; McLendon, C.; Liu, C.; Zhang, W.; Gerloff, D. L.; Huang, Z.; Tan, W.; Benner, S. A. J. Am. Chem. Soc. 2015, 137, 6734-6737. (13) Merritt, K. K.; Bradley, K. M.; Hutter, D.; Matsuura, M. F.; Rowold, D. J.; Benner, S. A. Beilstein Journal of Organic Chemistry 2014, 10, 2348-2360. (14) Rao, P.; Benner, S. A. The Journal of Organic Chemistry 2001, 66, 5012-5015. (15) Sismour, A. M.; Lutz, S.; Park, J.-H.; Lutz, M. J.; Boyer, P. L.; Hughes, S. H.; Benner, S. A. Nucleic Acids Res. 2004, 32, 728-735. (16) Matsuura, M. F.; Shaw, R. W.; Moses, J. D.; Kim, H.J.; Kim, M.-J.; Kim, M.-S.; Hoshika, S.; Karalkar, N.; Benner, S. A. ACS Synthetic Biology 2016, 5, 234-240. (17) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (18) Wilson, P. M.; LaBonte, M. J.; Russell, J.; Louie, S.; Ghobrial, A. A.; Ladner, R. D. Nucleic Acids Res. 2011, 39, e112.

Page 4 of 5

(19) Kutyavin, I. V.; Rhinehart, R. L.; Lukhtanov, E. A.; GornVv; Meyer, R. B.; Gamper, H. B. Biochemistry 1996, 35, 11170-11176. (20) Sintim, H. O.; Kool, E. T. J. Am. Chem. Soc. 2006, 128, 396-397. (21) Rao, T. V. S.; Haber, M. T.; Sayer, J. M.; Jerina, D. M. Bioorg. Med. Chem. Lett. 2000, 10, 907-910. (22) Sismour, A. M.; Benner, S. A. Nucleic Acids Res. 2005, 33, 5640-5646. (23) Lezius, A. G.; Scheit, K. H. European Journal of Biochemistry 1967, 3, 85-&. (24) Vormbroc.R; Morawiet.R; Gassen, H. G. Biochimica Et Biophysica Acta 1974, 340, 348-358. (25) Rackwitz, H. R.; Scheit, K. H. European Journal of Biochemistry 1977, 72, 191-200. (26) Krackov, M. H.; Lee, C. M.; Mautner, H. G. J. Am. Chem. Soc. 1965, 87, 892-896. (27) Hammes, G. G.; Park, A. C. J. Am. Chem. Soc. 1969, 91, 956-961. (28) Donohue, J. Journal of Molecular Biology 1969, 45, 231-235. (29) Laos, R.; Thomson, J. M.; Benner, S. A. Frontiers in Microbiology 2014, 5. (30) Marlière, P.; Patrouix, J.; Döring, V.; Herdewijn, P.; Tricot, S.; Cruveiller, S.; Bouzon, M.; Mutzel, R. Angewandte Chemie International Edition 2011, 50, 7109-7114. (31) Eremeeva, E.; Abramov, M.; Margamuljana, L.; Rozenski, J.; Pezo, V.; Marlière, P.; Herdewijn, P. Angewandte Chemie International Edition 2016, n/a-n/a. (32) Hamano-Takaku, F.; Iwama, T.; Saito-Yano, S.; Takaku, K.; Monden, Y.; Kitabatake, M.; Söll, D.; Nishimura, S. J. Biol. Chem. 2000, 275, 40324-40328. (33) Lajoie, M. J.; Söll, D.; Church, G. M. Journal of Molecular Biology 2016, 428, 1004-1021. (34) Petruska, J.; Goodman, M. F.; Boosalis, M. S.; Sowers, L. C.; Cheong, C.; Tinoco, I. Proceedings of the National Academy of Sciences of the United States of America 1988, 85, 6252-6256. (35) Reha-Krantz, L. J.; Hariharan, C.; Subuddhi, U.; Xia, S.; Zhao, C.; Beckman, J.; Christian, T.; Konigsberg, W. Biochemistry 2011, 50, 10136-10149. (36) Bande, O.; Braddick, D.; Agnello, S.; Jang, M.; Pezo, V.; Schepers, G.; Rozenski, J.; Lescrinier, E.; Marliere, P.; Herdewijn, P. Chem. Sci. 2016, 7, 995-1010. (37) Kirnos, M. D.; Khudyakov, I. Y.; Alexandrushkina, N. I.; Vanyushin, B. F. Nature 1977, 270, 369-370. (38) personal communication. (39) Marliere, P. In no. 20060270005 U.S.P Application, Ed. 2006.

ACS Paragon Plus Environment

Page 5 of 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

“For Table of Contents Use Only”

Polymerase Interactions with Wobble Mismatches in Synthetic Genetic Systems and their Evolutionary Implications Christian B. Winiger a, Myong-Jung Kima,b, Shuichi Hoshikaa,b, Ryan W. Shawa,b, Jennifer D. Mosesa,b, Mariko F. Matsuuraa,c, Dietlind L. Gerloffa, and Steven A. Bennera,b*

ACS Paragon Plus Environment

5