Single-turnover Kinetic Analysis of the Mutagenic Potential of 8-Oxo-7,8-dihydro-2′-deoxyguanosine during Gap-filling Synthesis Catalyzed by Human DNA Polymerases λ and β

Brown, Jessica A.; Duym, Wade W.; Fowler, Jason D.; Suo, Zucai

doi:10.1016/j.jmb.2007.01.069

Cited by 62 publications

(97 citation statements)

References 57 publications

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“…These findings suggest that 8-OH-dGTP was incorporated opposite A, and dCTP was then inserted opposite the 16 incorporated 8-OH-Gua residue during the next round of replication [26]. It was previously shown that mammalian DNA pols misinserted this oxidized dGTP opposite A in vitro [29,30,[40][41][42][43][44]. In particular, DNA pol η, a Y-family DNA pol, erroneously incorporated 8-OH-dGTP opposite A quite frequently [29,30].…”

Section: A:tc:g Transversion Mutations Were Induced By 8-oh-dgtp In mentioning

confidence: 96%

Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

et al. 2009

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In contrast, the knock-down of DNA polymerase ι did not affect the 8-OH-dGTP-induced mutations. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols η plus ζ and REV1 plus DNA pol ζ (but not by that of DNA pol η plus REV1),suggesting that REV1-DNA pol η and DNA pol ζ work in different steps.These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by the oxidized dGTP.3

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Section: A:tc:g Transversion Mutations Were Induced By 8-oh-dgtp In mentioning

confidence: 96%

Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

et al. 2009

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“…1). Polλ (13-16) fills DNA gaps and plays putative roles in base excision repair (13)(14)(15)(16)(17), nonhomologous end joining (18), and V(D)J recombination (19). Our structures illustrate how L-nucleotides, relative to D-dNTPs, are bound within the active site of a polymerase and then proceed through catalysis, and also facilitate the development of less toxic and more potent antiviral L-nucleoside analogs.…”

Section: Significancementioning

confidence: 99%

Structural basis for the binding and incorporation of nucleotide analogs with L -stereochemistry by human DNA polymerase λ

Vyas¹,

Zahurancik

Suo

2014

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

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Although lamivudine and emtricitabine, two L-deoxycytidine analogs, have been widely used as antiviral drugs for years, a structural basis for D-stereoselectivity against L-dNTPs, enantiomers of natural nucleotides (D-dNTPs), by any DNA polymerase or reverse transcriptase has not been established due to lack of a ternary structure of a polymerase, DNA, and an incoming L-dNTP. Here, we report 2.10-2.25 Å ternary crystal structures of human DNA polymerase λ, DNA, and L-deoxycytidine 5′-triphosphate (L-dCTP), or the triphosphates of lamivudine ((−)3TC-TP) and emtricitabine ((−)FTC-TP) with four ternary complexes per asymmetric unit. The structures of these 12 ternary complexes reveal that relative to D-deoxycytidine 5′-triphosphate (D-dCTP) in the canonical ternary structure of Polλ-DNA-D-dCTP, L-dCTP, (−)3TC-TP, and (−)FTC-TP all have their ribose rotated by 180°. Among the four ternary complexes with a specific L-nucleotide, two are similar and show that the L-nucleotide forms three Watson-Crick hydrogen bonds with the templating nucleotide dG and adopts a chair-like triphosphate conformation. In the remaining two similar ternary complexes, the L-nucleotide surprisingly interacts with the side chain of a conserved active site residue R517 through one or two hydrogen bonds, whereas the templating dG is anchored by a hydrogen bond with the side chain of a semiconserved residue Y505. Furthermore, the triphosphate of the L-nucleotide adopts an unprecedented N-shaped conformation. Our mutagenic and kinetic studies further demonstrate that the side chain of R517 is critical for the formation of the abovementioned four complexes along proposed catalytic pathways for L-nucleotide incorporation and provide the structural basis for the D-stereoselectivity of a DNA polymerase. (Fig. 1), possess L-stereochemistry. Both lamivudine and emtricitabine have been shown to be more effective in inhibiting HIV-1 RT and less toxic than their enantiomeric D-isomers (1-4). In addition, both lamivudine, a potent inhibitor of hepatitis B virus (HBV) (5), and telbivudine, the L-analog of thymidine, are approved drugs for the treatment of HBV infection, whereas emtricitabine is currently in clinical trials for this purpose (6). These L-nucleoside analogs demonstrate less clinical toxicity than their corresponding D-isomers, likely because human DNA polymerases possess strong D-stereoselectivity by preferentially binding and incorporating D-dNTPs over unnatural nucleotides with L-stereochemistry (L-dNTPs) during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by any DNA polymerase or RT has not been established, although D-stereoselectivity has been successfully explored in antiviral drug development.Despite high clinical efficacy, NRTIs are often associated with various drug toxicities resulting from the inhibition of host DNA polymerases that share a catalytic mechanism akin to HIV-1 RT (7). There are 16 identified human DNA polymerases that belong to A-, B-, X-, and Y-families. NRTI-associated mitoc...

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“…Most importantly, this type of plot readily exposes the reason for the loss in discrimination; i.e., why the vertical line gets shorter. For pol β, although dCTP insertion efficiency is reduced opposite 8-oxoG relative to guanine, the loss in discrimination is primarily due to the dramatic increase in catalytic efficiency of insertion of dATP opposite 8-oxoG (~4000-fold) [20]. This is probably due to the ambivalent coding properties of 8-oxoG (Fig.…”

Section: Influence Of 8-oxog On Dna Polymerase β Structure/functionmentioning

confidence: 99%

“…3, left panel) [20]. These ternary complex structures indicate that the templating 8-oxoG has assumed the syn -conformation and forms a Hoogsteen base pair with adenine.…”

Section: Influence Of 8-oxog On Dna Polymerase β Structure/functionmentioning

confidence: 99%

DNA polymerase structure-based insight on the mutagenic properties of 8-oxoguanine

Beard

Batra

Wilson

2010

Mutation Research/Genetic Toxicology and Environmental Mutagene

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An aerobic environment burdens DNA polymerase substrates with oxidized substrates (DNA and nucleotide pools). A major promutagenic lesion resulting from oxidative stress is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG). Guanine oxidation alters the hydrogen bonding properties of the base and glycosidic-preference of the nucleotide. The favored glycosidic syn-conformation exposes the Hoogsteen edge of the base for hydrogen bonding with adenine during DNA synthesis. The cell has recognized the threat of this lesion and has evolved an intricate surveillance system to provide DNA polymerases with unmodified substrates. Failure to do so leads to transversion mutations. Since the mutagenic properties of the base are dictated by the anti-syn conformation of the nucleotide, the molecular interactions of 8-oxoG in the confines of the DNA polymerase active site are expected to influence its coding potential. Recent structural characterization of DNA polymerases from several families with this lesion in the nascent base pair binding pocket has provided insight to the mutagenic properties of this modified nucleotide. These structures reveal that flexibility around the template binding pocket can permit 8-oxoG to assume an anti-or syn-conformation and code for cytosine or adenine incorporation, respectively. In contrast, the binding pocket for the incoming nucleotide does not have this flexibility so that 8-oxodGTP insertion opposite cytosine is strongly discouraged.

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Single-turnover Kinetic Analysis of the Mutagenic Potential of 8-Oxo-7,8-dihydro-2′-deoxyguanosine during Gap-filling Synthesis Catalyzed by Human DNA Polymerases λ and β

Cited by 62 publications

References 57 publications

Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

Structural basis for the binding and incorporation of nucleotide analogs with L -stereochemistry by human DNA polymerase λ

DNA polymerase structure-based insight on the mutagenic properties of 8-oxoguanine

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