Enzymatic bypass and the structural basis of miscoding opposite the DNA adduct 1,N2-ethenodeoxyguanosine by human DNA translesion polymerase η.

Etheno (ε)-adducts, e.g., 1,N2-ε-guanine (1,N2-ε-G) and 1,N6-ε-adenine (1,N6-ε-A), are formed through the reaction of DNA with metabolites of vinyl compounds or with lipid peroxidation products. These lesions are known to be mutagenic, but it is unknown how they lead to errors in DNA replication that are bypassed by DNA polymerases. Here we report the structural basis of misincorporation frequencies across from 1,N2-ε-G by human DNA polymerase (hpol) η. In single-nucleotide insertions opposite the adduct 1,N2-ε-G, hpol η preferentially inserted dGTP, followed by dATP, dTTP, and dCTP. This preference for purines was also seen in the first extension step. Analysis of full-length extension products by LC-MS/MS revealed that G accounted for 85% of nucleotides inserted opposite 1,N2-ε-G in single base insertion, and 63% of bases inserted in the first extension step. Extension from the correct nucleotide pair (C) was not observed, but the primer with A paired opposite 1,N2-ε-G was readily extended. Crystal structures of ternary hpol η insertion-stage complexes with nonhydrolyzable nucleotides dAMPnPP or dCMPnPP showed a syn orientation of the adduct, with the incoming A staggered between adducted base and the 5'-adjacent T, while the incoming C and adducted base were roughly coplanar. The formation of a bifurcated H-bond between incoming dAMPnPP and 1,N2-ε-G and T, compared with the single H-bond formed between incoming dCMPnPP and 1,N2-ε-G, may account for the observed facilitated insertion of dGTP and dATP. Thus, preferential insertion of purines by hpol η across from etheno adducts contributes to distinct outcomes in error-prone DNA replication.

Structural and Kinetic Analysis of Miscoding Opposite the DNA Adduct 1,N6-Ethenodeoxyadenosine by Human Translesion DNA Polymerase η.

1,N(6)-Ethenodeoxyadenosine (1,N(6)-ϵdA) is the major etheno lesion formed in the reaction of DNA with epoxides substituted with good leaving groups (e.g. vinyl chloride epoxide). This lesion is also formed endogenously in DNA from lipid oxidation. Recombinant human DNA polymerase η (hpol η) can replicate oligonucleotide templates containing 1,N(6)-ϵdA. In steady-state kinetic analysis, hpol η preferred to incorporate dATP and dGTP, compared with dTTP. Mass spectral analysis of incorporation products also showed preferred purine (A, G) incorporation and extensive -1 frameshifts, suggesting pairing of the inserted purine and slippage before further replication. Five x-ray crystal structures of hpol η ternary complexes were determined, three at the insertion and two at the extension stage. Two insertion complexes revealed incoming non-hydrolyzable dATP or dGTP analogs not pairing with but instead in a staggered configuration relative to 1,N(6)-ϵdA in the anti conformation, thus opposite the 5'-T in the template, explaining the proclivity for frameshift misincorporation. In another insertion complex, dTTP was positioned opposite 1,N(6)-ϵdA, and the adduct base was in the syn conformation, with formation of two hydrogen bonds. At the extension stage, with either an incorporated dA or dT opposite 1,N(6)-ϵdA and 2'-deoxythymidine-5'-[(α,ß)-imido]triphosphate opposite the 5'-A, the 3'-terminal nucleoside of the primer was disordered, consistent with the tendency not to incorporate dTTP opposite 1,N(6)-ϵdA. Collectively, the results show a preference for purine pairing opposite 1,N(6)-ϵdA and for -1 frameshifts.

Roles of Residues Arg-61 and Gln-38 of Human DNA Polymerase η in Bypass of Deoxyguanosine and 7,8-Dihydro-8-oxo-2'-deoxyguanosine.

Like the other Y-family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo.

Mechanism of Error-Free Bypass of the Environmental Carcinogen N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone Adduct by Human DNA Polymerase†η.

The environmental pollutant 3-nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8-dG-ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error-free manner by the human Y-family polymerase η (hPol η). Here, we report the crystal structure of a ternary Pol⋅DNA⋅dCTP complex between a C8-dG-ABA-containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error-free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra-nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson-Crick pair with the incoming dCTP.

Kinetics, structure, and mechanism of 8-Oxo-7,8-dihydro-2'-deoxyguanosine bypass by human DNA polymerase η.

DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion.

Structural Basis for Error-Free Bypass of the 5-N-Methylformamidopyrimidine-dG Lesion by Human DNA Polymerase η and Sulfolobus solfataricus P2 Polymerase IV.

N(6)-(2-Deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) arises from N7-methylation of deoxyguanosine followed by imidazole ring opening. The lesion has been reported to persist in animal tissues. Previous in vitro replication bypass investigations of the MeFapy-dG adduct revealed predominant insertion of C opposite the lesion, dependent on the identity of the DNA polymerase (Pol) and the local sequence context. Here we report crystal structures of ternary Pol·DNA·dNTP complexes between MeFapy-dG-adducted DNA template:primer duplexes and the Y-family polymerases human Pol η and P2 Pol IV (Dpo4) from Sulfolobus solfataricus. The structures of the hPol η and Dpo4 complexes at the insertion and extension stages, respectively, are representative of error-free replication, with MeFapy-dG in the anti conformation and forming Watson-Crick pairs with dCTP or dC.

Structure, stability and function of 5-chlorouracil modified A:U and G:U base pairs.

The thymine analog 5-chlorouridine, first reported in the 1950s as anti-tumor agent, is known as an effective mutagen, clastogen and toxicant as well as an effective inducer of sister-chromatid exchange. Recently, the first microorganism with a chemically different genome was reported; the selected Escherichia coli strain relies on the four building blocks 5-chloro-2'-deoxyuridine (ClU), A, C and G instead of the standard T, A, C, G alphabet [Marlière,P., Patrouix,J., Döring,V., Herdewijn,P., Tricot,S., Cruveiller,S., Bouzon,M. and Mutzel,R. (2011) Chemical evolution of a bacterium's genome. Angew. Chem. Int. Ed., 50, 7109-7114]. The residual fraction of T in the DNA of adapted bacteria was <2% and the switch from T to ClU was accompanied by a massive number of mutations, including >1500 A to G or G to A transitions in a culture. The former is most likely due to wobble base pairing between ClU and G, which may be more common for ClU than T. To identify potential changes in the geometries of base pairs and duplexes as a result of replacement of T by ClU, we determined four crystal structures of a B-form DNA dodecamer duplex containing ClU:A or ClU:G base pairs. The structures reveal nearly identical geometries of these pairs compared with T:A or T:G, respectively, and no consequences for stability and cleavage by an endonuclease (EcoRI). The lack of significant changes in the geometry of ClU:A and ClU:G base pairs relative to the corresponding native pairs is consistent with the sustained unlimited self-reproduction of E. coli strains with virtually complete T→ClU genome substitution.

High fidelity base pairing at the 3'-terminus.

Binding target strands with single base selectivity at a terminal position is difficult with natural DNA or RNA hybridization probes. Nature uses a degenerate genetic code that is based on RNA:RNA codon:anticodon duplexes tolerating wobble base pairs at the terminus. The importance of short RNA strands in regulatory processes in the cell make it desirable to develop receptor-like approaches for high fidelity binding, even at the very 3'-terminus of a probe. Here, we report the three-dimensional structure of a DNA duplex with a 3'-terminal 2'-anthraquinoylamido-2'-deoxyuridine (Uaq) residue that was solved by NMR and restrained molecular dynamics. The Uaq residue binds the 5'-terminus of the target strand through a combination of pi-stacking, hydrogen bonding, and interactions in the minor groove. The acylated aminonucleoside is the best molecular cap for 3'-termini reported to date. The Uaq motif assists binding of DNA strands, but is particularly effective in enhancing the affinity for RNA target strands, with a DeltaT(m) in the UV melting point of up to +18.2 degrees C per residue. Increased base pairing selectivity is induced for all sequence motifs tested, even in cases where unmodified duplexes show no preference for the canonical base pair at all. A single mismatched nucleobase facing the 3'-terminus gives DeltaDeltaT(m) values as large as -23.9 degrees C (RNA) or -29.5 degrees C (DNA). The 5'-phosphoramidite of the Uaq cap reported here allows for routine incorporation during automated syntheses.

Immunostimulatory CpG oligonucleotides form defined three-dimensional structures: results from an NMR study.

The DNA eicosamer 5'-TCCATGACGTTCCTGATGCT-3' is known to stimulate the innate immune system of vertebrae. The immunostimulatory activity is based on the activation of Toll-like receptor 9 (TLR9). While it is known that the CG dinucleotide of the eicosamer has to be unmethylated, the structural basis of the recognition of the DNA through the receptor remains unclear. Oligodeoxynucleotides containing the sequence of the eicosamer, or a portion thereof, ranging in length from hexamer to pentaeicosamer were studied by (1)H NMR spectroscopy. Based on two-dimensional NMR spectra, a number of resonances could be unambiguously assigned. For all oligonucleotides, structural transitions were detected upon heating, as monitored by the line width and chemical shift of low-field resonances. This includes the TC dinucleotide of the 5'-terminal portion, which does not have any clear base-pairing partners. The melting transitions, together with the NOESY cross-peaks, demonstrate that structure formation occurs well beyond the core hexamer 5'-GACGTT-3', a fact that may be important for understanding the molecular recognition by the Toll-like receptors of the innate immune system.