Structural Insights into the Specificity of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Family X DNA Polymerases

8-oxo-guanine (8OG) is a common base lesion, generated by reactive oxygen species, which has been associated with human diseases such as cancer, aging-related neurodegenerative disorders and atherosclerosis. 8OG is highly mutagenic, due to its dual-coding potential it can pair both with adenine or cytidine. Therefore, it creates a challenge for DNA polymerases striving to correctly replicate and/or repair genomic or mitochondrial DNA. Numerous structural studies provide insights into the mechanistic basis of the specificity of 8OG bypass by DNA polymerases from different families. Here, we focus on how repair polymerases from Family X (Pols β, λ and µ) engage DNA substrates containing the oxidized guanine. We review structures of binary and ternary complexes for the three polymerases, which represent distinct steps in their catalytic cycles—the binding of the DNA substrate and the incoming nucleotide, followed by its insertion and extension. At each of these steps, the polymerase may favor or exclude the correct C or incorrect A, affecting the final outcome, which varies depending on the enzyme.

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Promutagenic bypass of 7,8-dihydro-8-oxoadenine by translesion synthesis DNA polymerase Dpo4

Biochemical Journal ◽

10.1042/bcj20200449 ◽

2020 ◽

Author(s):

Hunmin Jung ◽

Seongmin Lee

Keyword(s):

Base Pair ◽

Dna Polymerases ◽

Translesion Synthesis ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Dual Coding ◽

Base Pairing ◽

Anti Conformation ◽

Hoogsteen Base Pairing ◽

Coding Potential

Reactive oxygen species induced by ionizing radiation and metabolic pathways generate 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as two major forms of oxidative damage. The mutagenicity of oxoG, which promotes G to T transversions, is attributed to the lesion’s conformational flexibility that enables Hoogsteen base pairing with dATP in the confines of DNA polymerases. The mutagenesis mechanism of oxoA, which preferentially causes A to C transversions, remains poorly characterized. While structures for oxoA bypass by human DNA polymerases are available, that of prokaryotic DNA polymerases have not been reported. Herein, we report kinetic and structural characterizations of Sulfolobus solfataricus Dpo4 incorporating a nucleotide opposite oxoA. Our kinetic studies show oxoA at the templating position reduces the replication fidelity by ~560-fold. The catalytic efficiency of the oxoA:dGTP insertion is ~300-fold greater than that of the dA:dGTP insertion, highlighting the promutagenic nature of oxoA. The relative efficiency of the oxoA:dGTP misincorporation is ~5-fold greater than that of the oxoG:dATP misincorporation, suggesting the mutagenicity of oxoA is comparable to that of oxoG. In the Dpo4 replicating base pair site, oxoA in the anti-conformation forms a Watson-Crick base pair with an incoming dTTP, while oxoA in the syn-conformation assumes Hoogsteen base pairing with an incoming dGTP, displaying the dual coding potential of the lesion. Within the Dpo4 active site, the oxoA:dGTP base pair adopts a Watson-Crick-like geometry, indicating Dpo4 influences the oxoA:dGTP base pair conformation. Overall, the results reported here provide insights into the miscoding properties of the major oxidative adenine lesion during translesion synthesis.

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Snapshots of a modified nucleotide moving through the confines of a DNA polymerase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811518115 ◽

2018 ◽

Vol 115 (40) ◽

pp. 9992-9997 ◽

Cited By ~ 16

Author(s):

Heike Maria Kropp ◽

Simon Leonard Dürr ◽

Christine Peter ◽

Kay Diederichs ◽

Andreas Marx

Keyword(s):

Dna Polymerase ◽

Dna Polymerases ◽

Ternary Complexes ◽

Structural Basis ◽

Modified Nucleotides ◽

Molecular Mechanics Calculations ◽

Modified Dna ◽

Substrate Properties ◽

Dna Strand ◽

Dna Substrate

DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems.

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Structural Insights into the Processing of Nucleobase-Modified Nucleotides by DNA Polymerases

Accounts of Chemical Research ◽

10.1021/acs.accounts.5b00544 ◽

2016 ◽

Vol 49 (3) ◽

pp. 418-427 ◽

Cited By ~ 92

Author(s):

Audrey Hottin ◽

Andreas Marx

Keyword(s):

Dna Polymerases ◽

Modified Nucleotides ◽

Structural Insights

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Incorporation of Oxidized Guanine Nucleoside 5′-Triphosphates in DNA with DNA Polymerases and Preparation of Single-Lesion Carrying DNA†

Biochemistry ◽

10.1021/bi7022199 ◽

2008 ◽

Vol 47 (16) ◽

pp. 4788-4799 ◽

Cited By ~ 5

Author(s):

Sophie Mourgues ◽

Jérôme Trzcionka ◽

Jean-Jacques Vasseur ◽

Geneviève Pratviel ◽

Bernard Meunier

Keyword(s):

Dna Polymerases ◽

Oxidized Guanine ◽

Single Lesion

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Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713027922 ◽

2014 ◽

Vol 70 (2) ◽

pp. 354-361 ◽

Cited By ~ 10

Author(s):

Rob Abdur ◽

Oksana O. Gerlits ◽

Jianhua Gan ◽

Jiansheng Jiang ◽

Jozef Salon ◽

...

Keyword(s):

Nucleic Acid ◽

Complex Structure ◽

Rnase H ◽

Acid Complex ◽

Mad Phasing ◽

Sad Phasing ◽

Protein Nucleic Acid ◽

Dna Complex ◽

Dna Substrate ◽

Structural Insights

The crystal structures of protein–nucleic acid complexes are commonly determined using selenium-derivatized proteinsviaMAD or SAD phasing. Here, the first protein–nucleic acid complex structure determined using selenium-derivatized nucleic acids is reported. The RNase H–RNA/DNA complex is used as an example to demonstrate the proof of principle. The high-resolution crystal structure indicates that this selenium replacement results in a local subtle unwinding of the RNA/DNA substrate duplex, thereby shifting the RNA scissile phosphate closer to the transition state of the enzyme-catalyzed reaction. It was also observed that the scissile phosphate forms a hydrogen bond to the water nucleophile and helps to position the water molecule in the structure. Consistently, it was discovered that the substitution of a single O atom by a Se atom in a guide DNA sequence can largely accelerate RNase H catalysis. These structural and catalytic studies shed new light on the guide-dependent RNA cleavage.

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Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase

The EMBO Journal ◽

10.1038/sj.emboj.7600354 ◽

2004 ◽

Vol 23 (17) ◽

pp. 3452-3461 ◽

Cited By ~ 151

Author(s):

Luis G Brieba ◽

Brandt F Eichman ◽

Robert J Kokoska ◽

Sylvie Doublié ◽

Tom A Kunkel ◽

...

Keyword(s):

Dna Polymerase ◽

Dual Coding ◽

Structural Basis ◽

High Fidelity ◽

Coding Potential

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Structure of DNA Polymerase β with the Mutagenic DNA Lesion 8-Oxodeoxyguanine Reveals Structural Insights into Its Coding Potential

Structure ◽

10.1016/s0969-2126(02)00930-9 ◽

2003 ◽

Vol 11 (1) ◽

pp. 121-127 ◽

Cited By ~ 96

Author(s):

Joseph M Krahn ◽

William A Beard ◽

Holly Miller ◽

Arthur P Grollman ◽

Samuel H Wilson

Keyword(s):

Dna Polymerase ◽

Dna Polymerase Β ◽

Dna Lesion ◽

Structural Insights ◽

Coding Potential

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Structural insights into distinct signaling profiles of the μOR activated by diverse agonists

10.1101/2021.12.07.471645 ◽

2021 ◽

Author(s):

Qianhui Qu ◽

Weijiao Huang ◽

Deniz Aydin ◽

Joseph M. Paggi ◽

Alpay B. Seven ◽

...

Keyword(s):

G Protein ◽

Respiratory Depression ◽

Animal Studies ◽

Md Simulations ◽

Active State ◽

Μ Opioid Receptor ◽

Mechanistic Basis ◽

G Protein Coupled ◽

Structural Insights ◽

Different Parts

AbstractDrugs targeting the G protein-coupled μ-opioid receptor (μOR) are the most effective analgesics available but are also associated with fatal respiratory depression. While some partial opioid agonists appear to be safer than full agonists, the signaling pathways responsible for respiratory depression have yet to be elucidated. Here we investigated the structural and mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two μOR agonists with different safety profiles. LFT, one of the most potent and lethal opioids, and MP, a derivative from the kratom plant with reduced respiratory depression in animal studies at equianalgesic doses, exhibited markedly different signaling efficacy profiles for G protein subtype activation and recruitment of β-arrestins. Cryo-EM structures of the μOR-Gi1 complex with MP (2.5Å) and LFT (3.2Å) revealed that the two ligands engage distinct sub-pockets, and molecular dynamics (MD) simulations showed additional differences in the binding site that propagate to the intracellular side of the receptor where G proteins and β-arrestins bind. While MP favors the precise G protein-bound active state observed in the cryo-EM structures, LFT favors a distinct active state. These results highlight how drugs engaging different parts of the μOR orthosteric pocket can lead to distinct signaling outcomes.

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