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Computational Evaluation of Nucleotide Insertion Opposite Expanded and Widened DNA by the Translesion Synthesis Polymerase Dpo4

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Computational Evaluation of Nucleotide Insertion Opposite Expanded and Widened DNA by the Translesion Synthesis Polymerase Dpo4 molecules Article Computational Evaluation of Nucleotide Insertion Opposite Expanded and Widened DNA by the Translesion Synthesis Polymerase Dpo4 Laura Albrecht †, Katie A. Wilson † and Stacey D. Wetmore * Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge Alberta, AB T1K 3M4, Canada; [email protected] (L.A.); [email protected] (K.A.W.) * Correspondence: [email protected]; Tel.: +1-403-329-2323; Fax: +1-403-329-2057 † These authors contributed equally to this work. Academic Editors: James W. Gauld and Leif A. Eriksson Received: 16 May 2016; Accepted: 14 June 2016; Published: 23 June 2016 Abstract: Expanded (x) and widened (y) deoxyribose nucleic acids (DNA) have an extra benzene ring incorporated either horizontally (xDNA) or vertically (yDNA) between a natural pyrimidine base and the deoxyribose, or between the 5- and 6-membered rings of a natural purine. Far-reaching applications for (x,y)DNA include nucleic acid probes and extending the natural genetic code. Since modified nucleobases must encode information that can be passed to the next generation in order to be a useful extension of the genetic code, the ability of translesion (bypass) polymerases to replicate modified bases is an active area of research. The common model bypass polymerase DNA polymerase IV (Dpo4) has been previously shown to successfully replicate and extend past a single modified nucleobase on a template DNA strand. In the current study, molecular dynamics (MD) simulations are used to evaluate the accommodation of expanded/widened nucleobases in the Dpo4 active site, providing the first structural information on the replication of (x,y)DNA. Our results indicate that the Dpo4 catalytic (palm) domain is not significantly impacted by the (x,y)DNA bases. Instead, the template strand is displaced to accommodate the increased C1’–C1’ base-pair distance. The structural insights unveiled in the present work not only increase our fundamental understanding of Dpo4 replication, but also reveal the process by which Dpo4 replicates (x,y)DNA, and thereby will contribute to the optimization of high fidelity and efficient polymerases for the replication of modified nucleobases. Keywords: expanded DNA; xDNA; widened DNA; yDNA; DNA replication; translesion synthesis; bypass polymerase; Dpo4; molecular dynamics 1. Introduction At the cellular level, living organisms store and process genetic information encoded by a set of four DNA nucleobases (A, C, G, and T), which combine according to Watson-Crick hydrogen-bonding rules. There have been many attempts to expand the unique function and applications of DNA [1,2]. Approaches considered to date include editing the DNA backbone (e.g., polyamide nucleic acids (PNA), locked nucleic acids (LNA), and xeno-nucleic acids (XNA) [3–5]), modifying the nucleobase functional groups (e.g., difluorotoluene, isoguanine, and 2,6-diaminopurine [6–9]), or changing the underlying nucleobase composition (e.g., propynyl isocarbostyril and fleximers [10–12]). In addition to exploring fundamental questions about nature’s building blocks, modified DNA has many practical applications, including DNA nanomaterials [13,14], therapeutic approaches such as antiviral drugs or the delivery of genetic material to cells [15–19], nucleic acid computing [20–23], and tools to probe the mechanisms of biological processes [24]. Molecules 2016, 21, 822; doi:10.3390/molecules21070822 www.mdpi.com/journal/molecules Molecules 2016, 21, 822 2 of 22 Molecules 2016, 21, 822 2 of 22 One classclass ofof modified modified DNA DNA that that shows shows promising promising applications applications both asboth an extensionas an extension of the naturalof the geneticnatural codegenetic and code as a nucleicand as acida nucleic probe acid is expanded probe is (x) expanded and widened (x) and (y) DNA.widened These (y) modificationsDNA. These havemodifications a single benzene have a single spacer benzene incorporated spacer horizontally incorporated (xDNA) horizontally or vertically (xDNA) (yDNA) or vertically either between(yDNA) aeither natural between pyrimidine a natural base pyrimidine and deoxyribose, base and ordeox betweenyribose, the or 5-between and 6-membered the 5- and 6-membered rings of a natural rings purineof a natural (Figure purine1). Although (Figure 1). base Although extension base increases extension the increases base-pair the C1 base-pair1–C1’ distance, C1′–C1’ the distance, canonical the Watson-Crickcanonical Watson-Crick hydrogen-bonding hydrogen-b facesonding are maintained faces are maintained in (x,y)DNA in nucleobases,(x,y)DNA nucleobases, which allows which for complementaryallows for complementary base pairing. base The pairing. first benzene-ring The first benzene-ring expanded nucleobasesexpanded nucleobases were RNA were analogues RNA proposedanalogues byproposed Leonard by and Leonard coworkers and coworkers [25–27]. The [25–27]. Kool The group Kool has group since has synthesized since synthesized a series ofa expandedseries of expanded and widened and DNAwidened nucleobases DNA nucleobases [28–35]. Theoretical [28–35]. Theoretical and experimental and experimental studies show studies that helicesshow that composed helices composed of (x,y)DNA:DNA of (x,y)DNA:DNA base pairs base are pairs overall are more overall stable more than stable natural than B-DNAnatural B-DNA [36,37], at[36,37], least at in least part in due part to due enhanced to enhanced stacking stacking [38], [38], as well as well as exhibitas exhibit increased increased charge-transfer charge-transfer [[39–41]39–41] and fluorescentfluorescent [[36,42–45]36,42–45] properties.properties. Compar Compareded to to B-DNA, mixed xDNA–DNA helices havehave increasedincreased majormajor andand minorminor groovegroove widths,widths, andand aa reducedreduced helicalhelical twist,twist, whilewhile mixedmixed yDNA–DNAyDNA–DNA helices have increased base-pair inclination, reduced twist, and smaller major and minorminor groovegroove widthswidths [[34–36,41,42,46–49].34–36,41,42,46–49]. Figure 1. Chemical structures of the DNA, xDNA and yDNA bases considered in the current study. In additionaddition toto thethe capacitycapacity to to store store information information through through complementary complementary pairing pairing in in stable stable helices, helices, it mustit must be possiblebe possible to replicate to replicate (x,y)DNA (x,y)DNA strands strands in order in for order these for modifications these modifications to be useful to extensionsbe useful ofextensions the natural of the genetic natural code. genetic In biological code. In systems,biological DNA systems, is copied DNA by is eithercopied standard by either replication standard orreplication translesion or (bypass)translesion synthesis (bypass) [50 ].synthesis Standard [5 replication0]. Standard uses replication replicative DNAuses polymerasesreplicative DNA that havepolymerases high fidelity, that have processivity high fidelity, and efficiency processivity for canonical and efficiency nucleotides, for canonical but are nucleotides, generally unable but are to replicategenerally non-standardunable to replicate (e.g., damagednon-standard or otherwise (e.g., damaged modified) or otherwise bases [51 ,52modifi]. Oned) the bases other [51,52]. hand, bypassOn the polymerasesother hand, bypass exploit polymerases a highly flexible exploit activea highly site flexible to replicate active site past to many replicate non-standard past many non-standard bases, albeit withbases, lower albeit fidelity, with lower processivity fidelity, and processivity efficiency compared and efficiency to replication compared of canonicalto replication DNA of by canonical standard polymerasesDNA by standard [50]. With polymerases the goal to improve[50]. With the the efficiency goal to of improve modified the DNA efficiency replication, of severalmodified research DNA groupsreplication, have several focused research on engineering groups novelhave focused polymerase on engineering variants (by novel modifying polymerase individual variants residues (by and/ormodifying domains) individual that residues exploit the and/or flexibility domains) of bypass that exploit polymerases the flexibility to replicate of bypass many polymerases non-standard to basesreplicate with many a range non-standard of biotechnological bases with applications a range of [biotechnological53–55]. applications [53–55]. Since the design of an (x,y)DNA replicase is anticipated to be extremely challenging [33], [33], experimental studies have initially investigated the replication of (x,y)DNA by existing polymerases. This approachapproach was atat leastleast inin partpart fuelledfuelled byby thethe successfulsuccessful replicationreplication ofof DNADNA containingcontaining expandedexpanded and widened widened nucleobases nucleobases in in EscherichiaEscherichia coli coli (E. (E.coli) coli) [56–58].[56– To58]. gain To more gain deta moreiled detailed information, information, kinetic kineticstudies studiesconsidered considered the replication the replication of a single of ax- singleor y-nucleotide x- or y-nucleotide (dxN or dyN) (dxN in or an dyN) otherwise in an otherwisecanonical DNA canonical duplex DNA by common duplex by model common replicative model (Klenow replicative fragment( (Klenowexo– fragment(), Kf(exo–exo))–), and Kf( bypassexo–)) and(Dpo4) bypass polymerases (Dpo4)polymerases [30,56,59]. These [30, 56studies,59]. Thesefurther studies confirmed further successful confirmed insertion successful of a complementary
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