Human DNA Primase Uses Watson−Crick Hydrogen Bonds To Distinguish between Correct and Incorrect Nucleoside Triphosphates

Moore, Chris; Živković, Aleksandra; Engels, Joachim W.; Kuchta, Robert D.

doi:10.1021/bi0490791

Cited by 31 publications

(37 citation statements)

References 27 publications

(70 reference statements)

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“…Human primase incorporates incorrect, natural NTPs at an high frequency (around 1 every 100 correct NTPs), yet very strongly discriminates against purine NTP analogues that cannot form a complete set of Watson-Crick hydrogen bonds (13). To better understand how the enzyme chooses whether or not to polymerize a NTP, we examined an expanded series of both purine and pyrimidine analogues when present in both the template and incoming NTP (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2010

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Human DNA primase synthesizes short RNA primers that DNA polymerase α 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-oxo-phenothiazine-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. KeywordsFidelity; misincorporation; hydrogen bonds; Watson-Crick; DNA polymerase; kinetics DNA polymerases lack the capacity to initiate the synthesis of new DNA strands de novo on single-stranded DNA templates. Rather, they require the 3' end of a pre-existing primer bound to the template in order to replicate the template. In most replicative systems, DNA primases solve this problem by synthesizing short RNA primers that a replicative DNA polymerase then elongates (1,2). In eukaryotes, primase synthesizes a short RNA primer (8 -12 nucleotides long) that DNA polymerase α (pol α) elongates by another about 20 nucleotides to generate a DNA primer(3). Pol α dissociates and either pol δ or pol ε finishes the bulk of DNA replication on the leading and lagging strands (4,5). After completion of Okazaki fragment synthesis on the lagging strand, the RNA primer is removed and replaced with DNA. Consequently, errors during primer synthesis do not become part of the genome and, therefore, primase need not † This work was supported by NIH grant GM54194 to R.D.K. and grants from the Academy of Sciences of the Czech Republic (Z4 055 905), the Ministry of Education (LC 512) and the Grant Agency of the ASCR (IAA400550902) to M. H.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2010

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“…5-Nitrobenzimidazole was purchased from Lancaster and 5-methylbenzimidazole and 5-methoxybenzimidazole were purchased from Sigma. 4-Trifluoromethyl-1H-benzimidazole, 5-trifluoromethyl-1H-benzimidazole (22) and 1-β- D -2′-deoxyribofuranosyl-(4-methylbenzimidazole)-5′-triphosphate (dZTP) (11) were synthesized as previously described.…”

Section: Methodsmentioning

confidence: 99%

Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase

Kincaid

Beckman²,

Živković³

et al. 2005

Nucleic Acids Research

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In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo−) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment.

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“…86 Several groups have made important contributions that have provided insights into this issue. 87−94 Kool’s group challenged Watson and Crick’s argument about the basis for nucleotide discrimination by using an array of nonpolar nucleoside analogues, such as 2′-deoxyribo-4-methylbenzimidazole (dZ), 2′-deoxyribo-2,4-difluorotoluene (dF), and 2′-deoxyribo-9-methyl-1 H -imidazole-[(4,5)-β]-pyridine (dQ), in primer extension assays to show that base pairing was not as important as shape for base selectivity. 86,95−97 The analogue that created the most controversy was dF, which was reported to be a nonpolar, non-hydrogen bonding isostere of dT.…”

Section: Factors That Contribute To the Dntp Insertion Efficiency Andmentioning

confidence: 99%

RB69 DNA Polymerase Structure, Kinetics, and Fidelity

Xia

Konigsberg

2014

Biochemistry

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This review will summarize our structural and kinetic studies of RB69 DNA polymerase (RB69pol) as well as selected variants of the wild-type enzyme that were undertaken to obtain a deeper understanding of the exquisitely high fidelity of B family replicative DNA polymerases. We discuss how the structures of the various RB69pol ternary complexes can be used to rationalize the results obtained from pre-steady-state kinetic assays. Our main findings can be summarized as follows. (i) Interbase hydrogen bond interactions can increase catalytic efficiency by 5000-fold; meanwhile, base selectivity is not solely determined by the number of hydrogen bonds between the incoming dNTP and the templating base. (ii) Minor-groove hydrogen bond interactions at positions n – 1 and n – 2 of the primer strand and position n – 1 of the template strand in RB69pol ternary complexes are essential for efficient primer extension and base selectivity. (iii) Partial charge interactions among the incoming dNTP, the penultimate base pair, and the hydration shell surrounding the incoming dNTP modulate nucleotide insertion efficiency and base selectivity. (iv) Steric clashes between mismatched incoming dNTPs and templating bases with amino acid side chains in the nascent base pair binding pocket (NBP) as well as weak interactions and large gaps between the incoming dNTPs and the templating base are some of the reasons that incorrect dNTPs are incorporated so inefficiently by wild-type RB69pol. In addition, we developed a tC°–tCnitro Förster resonance energy transfer assay to monitor partitioning of the primer terminus between the polymerase and exonuclease subdomains.

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Human DNA Primase Uses Watson−Crick Hydrogen Bonds To Distinguish between Correct and Incorrect Nucleoside Triphosphates

Cited by 31 publications

References 27 publications

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

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

Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase

RB69 DNA Polymerase Structure, Kinetics, and Fidelity

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