Nicolas Joubert

Optimization of the Pyridyl Nucleobase Scaffold for Polymerase Recognition and Unnatural Base Pair Replication

As part of an effort to increase both the biological and biotechnological applications of DNA, we[1–5] and others[6–9] have explored the DNA polymerase-mediated replication of a wide range of unnatural base pairs. In our initial efforts we examined large, aromatic, unnatural nucleotides, both as self pairs of two identical nucleotides and heteropairs of different nucleotides.[1–5,10,11] While several of these unnatural base pairs are efficiently synthesized (i.e. by insertion of the unnatural dNTP opposite its partner in the template) by the exonuclease-deficient Klenow fragment of E. coli DNA polymerase I (Kf), none are efficiently extended (i.e. by continued primer elongation), most likely due to interstrand nucleobase intercalation and distortion of the primer terminus.[10] Thus, a range of nucleotides bearing smaller phenyl-based nucleobases that should be incapable of intercalation were explored, and several modifications that facilitate extension were identified.[1–4] Of these, aza-substitution at the 2 position (2Py, Figure 1) appears to be the only modification that facilitates self pair extension without significantly facilitating mispairing.[3]

Mechanisms by Which Human DNA Primase Chooses To Polymerize a Nucleoside Triphosphate

Human DNA primase synthesizes short RNA primers that DNA polymerase alpha then elongates during the initiation of all new DNA strands. Even though primase misincorporates NTPs at a relatively high frequency, this likely does not impact the final DNA product since the RNA primer is replaced with DNA. We used an extensive series of purine and pyrimidine analogues to provide further insights into the mechanism by which primase chooses whether or not to polymerize a NTP. Primase readily polymerized a size-expanded cytosine analogue, 1,3-diaza-2-oxophenothiazine NTP, across from a templating G but not across from A. The enzyme did not efficiently polymerize NTPs incapable of forming two Watson-Crick hydrogen bonds with the templating base with the exception of UTP opposite purine deoxyribonucleoside. Likewise, primase did not generate base pairs between two nucleotides with altered Watson-Crick hydrogen-bonding patterns. Examining the mechanism of NTP polymerization revealed that human primase can misincorporate NTPs via both template misreading and a primer-template slippage mechanism. Together, these data demonstrate that human primase strongly depends on Watson-Crick hydrogen bonds for efficient nucleotide polymerization, much more so than the mechanistically related herpes primase, and provide insights into the potential roles of primer-template stability and base tautomerization during misincorporation.

Herpes simplex virus-1 DNA primase: a remarkably inaccurate yet selective polymerase.

Herpes simplex virus-1 primase misincorporates the natural NTPs at frequencies of around one error per 30 NTPs polymerized, making it one of the least accurate polymerases known. We used a series of nucleotide analogues to further test the hypothesis that primase requires Watson-Crick hydrogen bond formation to efficiently polymerize a NTP. Primase could not generate base pairs containing a complete set of hydrogen bonds in an altered arrangement (isoguanine.isocytosine) and did not efficiently polymerize dNTPs completely incapable of forming Watson-Crick hydrogen bonds opposite templating bases incapable of forming Watson-Crick hydrogen bonds. Similarly, primase did not incorporate most NTPs containing hydrophobic bases incapable of Watson-Crick hydrogen bonding opposite natural template bases. However, 2-pyridone NTP and 4-methyl-2-pyridone NTP provided striking exceptions to this rule. The effects of removing single Watson-Crick hydrogen bonding groups from either the NTP or templating bases varied from almost no effect to completely blocking polymerization depending both on the parental base pair (G.C vs A.T/U) and which base pair of the growing primer (second, third, or fourth) was examined. Thus, primase does not absolutely need to form Watson-Crick hydrogen bonds to efficiently polymerize a NTP. Additionally, we found that herpes primase can misincorporate nucleotides both by misreading the template and by a primer-template slippage mechanism. The mechanistic and biological implications of these results are discussed.

Alkyne-Azide Click Chemistry Mediated Carbanucleosides Synthesis

Hitherto unknown 1,4-disubstituted-[1,2,3]-triazolo-4′,4′-dihydroxymethyl-3′-deoxy carbanucleosides were synthesized based on a “click approach.” Various alkynes were introduced on a key azido intermediate by the “click” 1,3-dipolar Huisgen cycloaddition. Their antiviral activities and cellular toxicities were evaluated on vaccinia virus. None of the synthesized compounds exhibited a significant antiviral activity.

B Family DNA Polymerases Asymmetrically Recognize Pyrimidines and Purines

We utilized a series of pyrimidine analogues modified at O(2), N-3, and N(4)/O(4) to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O(2) of a pyrimidine dNTP vastly decreased the level of incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O(2) from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N(4)/O(4) greatly impaired polymerization, both of the resulting dNTP analogues and of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results with regard to how polymerases discriminate between right and wrong dNTPs are discussed.

Study of different copper(I) catalysts for the "click chemistry" approach to carbanucleosides

We compare herein the scope of three copper (I) catalysts on the synthesis of various 1,4-disubstitued-1,2,3-triazolo-carbanucleosides through a microwave (and thermic) assisted Huisgen 1,3-dipolar cycloaddition. The tetrakis(acetonitrile)copper hexafluorophosphate ([Cu(CH 3 CN)4]PF 6 ), the imidazoline(mesythyl)copper bromide (Imes)CuBr, and the copper/copper sulfate Cu(0)/CuSO 4 (II) mixture have been chosen for this study. Their influence in a catalytic amount will be analyzed according to the substituent of the alkyne, the solvent, or the heating method.