Structural Basis for Promutagenicity of 8-Halogenated Guanine

Koag, Myong Chul; Min, Kyoung‐Wook; Lee, Seongmin

doi:10.1074/jbc.m113.537803

Cited by 5 publications

(22 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To gain insights into this, we obtained ternary structures of polβ in precatalytic complex with an incoming nonhydrolyzable dCMPNPP (hereafter dCTP*) to be paired with templating ClG with both Mg 2+ metal ions (Figure 3). The use of this nucleotide analog has been suggested to retain the same structure as dCTP [42,43,44]. This precatalytic ClG:dCTP*(Mg 2+ ) ternary structure was refined to 2.0 Å (Figure 3A and Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…Polβ was expressed and purified from Escherichia coli with minor modifications of the method described previously [38]. The binary polβ complex containing templating ClG in a single-nucleotide gapped DNA was prepared under conditions similar to those described previously [42]. Polβ was complexed with a single-nucleotide gapped DNA containing a 16-mer template (5′-CCGAC(ClG)GCGCATCAGC-3′), a complementary 10-mer primer (5′-GCTGATGCGC-3′), and a 5-mer downstream oligonucleotide (5′-pGTCGG-3′).…”

Section: Methodsmentioning

confidence: 99%

“…The resulting polβ-DNA complex was used to obtain binary and ternary complex crystals in the absence or presence of an incoming nucleotide, respectively. The ternary polβ-DNA complex co-crystals with nonhydrolyzable dNTP analogs paired with templating ClG in a single-nucleotide gap at the active site were grown in a solution containing 50 mM imidazole, pH 7.5, 14–23% PEG3400, and 350 mM sodium acetate as described previously [42]. Crystals were cryo-protected in mother liquor supplemented with 12% ethylene glycol and were flash-frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Promutagenicity of 8-Chloroguanine, A Major Inflammation-Induced Halogenated DNA Lesion

2019

Self Cite

View full text Add to dashboard Cite

Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2′-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase β (polβ) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polβ incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Promutagenicity of 8-Chloroguanine, A Major Inflammation-Induced Halogenated DNA Lesion

2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…Polβ was expressed and purified as described previously. 35 Polβ binary complex with a single-nucleotide gap opposite templating O6MeG was prepared using the same conditions described previously. 35 Polβ ternary complex was prepared by adding nonhydrolyzable dCMPNPP or dTMPNPP (5.0 mM, Jena Biosciences) to the mixture of the polβ gapped binary complex.…”

Section: Methodsmentioning

confidence: 99%

Metal-Dependent Conformational Activation Explains Highly Promutagenic Replication across O6-Methylguanine by Human DNA Polymerase β

Koag

Lee

2014

J. Am. Chem. Soc.

Self Cite

View full text Add to dashboard Cite

Human DNA polymerase β (polβ) inserts, albeit slowly, T opposite the carcinogenic lesion O6-methylguanine (O6MeG) ∼30-fold more frequently than C. To gain insight into this promutagenic process, we solved four ternary structures of polβ with an incoming dCTP or dTTP analogue base-paired with O6MeG in the presence of active-site Mg2+ or Mn2+. The Mg2+-bound structures show that both the O6MeG·dCTP/dTTP–Mg2+ complexes adopt an open protein conformation, staggered base pair, and one active-site metal ion. The Mn2+-bound structures reveal that, whereas the O6Me·dCTP–Mn2+ complex assumes the similar altered conformation, the O6MeG·dTTP–Mn2+ complex adopts a catalytically competent state with a closed protein conformation and pseudo-Watson–Crick base pair. On the basis of these observations, we conclude that polβ slows nucleotide incorporation opposite O6MeG by inducing an altered conformation suboptimal for catalysis and promotes mutagenic replication by allowing Watson–Crick-mode for O6MeG·T but not for O6MeG·C in the enzyme active site. The O6MeG·dTTP–Mn2+ ternary structure, which represents the first structure of mismatched polβ ternary complex with a closed protein conformation and coplanar base pair, the first structure of pseudo-Watson–Crick O6MeG·T formed in the active site of a DNA polymerase, and a rare, if not the first, example of metal-dependent conformational activation of a DNA polymerase, indicate that catalytic metal-ion coordination is utilized as a kinetic checkpoint by polβ and is crucial for the conformational activation of polβ. Overall, our structural studies not only explain the promutagenic polβ catalysis across O6MeG but also provide new insights into the replication fidelity of polβ.

show abstract

“…Damaged substrates complicate the ability of DNA polymerases to select the correct nucleotide 23 - 25 . Structural studies have identified that correct and incorrect non-damaged nucleotides are discriminated from each other based on proper alignment of catalytic atoms.…”

mentioning

confidence: 99%

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

Freudenthal

Beard

Perera

et al. 2014

Nature

142

246

View full text Add to dashboard Cite

Oxidative stress promotes genomic instability and human diseases1. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2’-deoxyguanosine found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP)2,3. Nucleotide pools are especially vulnerable to oxidative damage4. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival5,6 and to modulate E. coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner7. How polymerase discriminates between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics8. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis9,10. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine (Cy) and 8-oxodGTP(syn) utilizes its Hoogsteen edge to base pair with adenine (Ad)11. Here we utilized time-lapse crystallography to follow 8-oxo-dGTP insertion opposite Ad or Cy with human DNA pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxodGTP utilizes charge modulation during insertion that can lead to a blocked DNA repair intermediate.

show abstract

Structural Basis for Promutagenicity of 8-Halogenated Guanine

Cited by 5 publications

References 48 publications

Promutagenicity of 8-Chloroguanine, A Major Inflammation-Induced Halogenated DNA Lesion

Promutagenicity of 8-Chloroguanine, A Major Inflammation-Induced Halogenated DNA Lesion

Metal-Dependent Conformational Activation Explains Highly Promutagenic Replication across O6-Methylguanine by Human DNA Polymerase β

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

Contact Info

Product

Resources

About