Mechanism of error-free and semitargeted mutagenic bypass of an aromatic amine lesion by Y-family polymerase Dpo4

Rechkoblit, Olga; Kolbanovskiy, Alexander; Malinina, Lucy; Geacintov, Nicholas E.; Broyde, Suse; Patel, Dinshaw J.

doi:10.1038/nsmb.1771

Cited by 45 publications

(92 citation statements)

References 59 publications

(103 reference statements)

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“…Exclusion of one or more unpaired template nucleotides from the DNA double-helix during the lesion bypass and/or extension steps will result in frameshift mutations. 40 The binding conformation of an excluded bulky adduct can sometimes be stabilized by its interactions with active site residues but will hinder DNA translocation during DNA polymerization. 45 Relative to the ternary structures with undamaged DNA, the aforementioned bulky lesions more or less affect the geometry of the polymerase active site and thereby decrease nucleotide incorporation efficiency (Table 3).…”

Section: Discussionmentioning

confidence: 99%

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

Vyas¹,

Efthimiopoulos²,

Tokarsky³

et al. 2015

J. Am. Chem. Soc.

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1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.

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Section: Discussionmentioning

confidence: 99%

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

Vyas¹,

Efthimiopoulos²,

Tokarsky³

et al. 2015

J. Am. Chem. Soc.

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“…While the nonacetylated lesions exist both in syn and anti conformation (14, 15), the corresponding acetylated lesions seem to adopt the syn-conformation with high preference (10,16,17). Crystal structures of Ellenberger, Beese, and Patel, showing the nonacetylated AF-dG-lesion inside different polymerases, prove that the lesion is indeed bound in anti conformation, allowing Watson-Crick base pairing with an incoming dCTP (18)(19)(20). While bypass of the nonacetylated lesions by high-fidelity polymerases is rather well understood, the mechanism that allows low-fidelity polymerases such as Pol η to replicate through acetylated AAF-dG lesions is still unknown.…”

mentioning

confidence: 99%

Mechanism of replication blocking and bypass of Y-family polymerase η by bulky acetylaminofluorene DNA adducts

Schorr

Schneider²,

Lammens³

et al. 2010

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

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Heterocyclic aromatic amines produce bulky C8 guanine lesions in vivo, which interfere and disrupt DNA and RNA synthesis. These lesions are consequently strong replication blocks. In addition bulky adducts give rise to point and frameshift mutations. The translesion synthesis (TLS) DNA polymerase η is able to bypass slowly C8 bulky adduct lesions such as the widely studied 2-aminofluorene-dG and its acetylated analogue mainly in an error-free manner. Replicative polymerases are in contrast fully blocked by the acetylated lesion. Here, we show that TLS efficiency of Pol η depends critically on the size of the bulky adduct forming the lesion. Based on the crystal structure, we show why the bypass reaction is so difficult and we provide a model for the bypass reaction. In our model, TLS is accomplished without rotation of the lesion into the anti conformation as previously thought.DNA damage | DNA polymerase | translesion DNA synthesis | aromatic amines A romatic amines are known to be strong carcinogens. After metabolic activation, aromatic amines react as electrophilic arylnitrenium ions with nucleophilic functionalities of the DNA duplex. Preferred reaction sides are the amino groups of adenine and guanine and particularly the C8-position of guanine (1, 2). The latter reaction gives rise to the so called C8-bulky adduct lesions, which interfere with the replication process leading to mutations (2). Two types of aromatic amine lesions are known. The nonacetylated lesions depicted in Fig. 1A reduce the replication efficiency but are in general faithfully bypassed by highfidelity polymerases (3). In contrast, the acetylated derivatives ( Fig. 1B) block replicative polymerases (3, 4), but can be bypassed with special low-fidelity polymerases (5-9). Moreover, if these acetylated lesions are present in certain repetitive sequences, they are known to induce frameshift mutations (4,(10)(11)(12). Recently, in vivo experiments revealed that the low-fidelity Y-family polymerase η is able to replicate DNA containing acetylated aromatic amine lesions such as the acetylated analogue of 2-aminofluorene-dG (AF-dG), 2-acetylaminofluorene-dG (AAF-dG) (13). Replication through the lesion is even for Pol η very difficult and hence slow, but if it occurs it is typically error free.The distinct mutagenic properties of the acetylated and nonacetylated aromatic amine lesions are caused by their different conformational preferences. While the nonacetylated lesions exist both in syn and anti conformation (14, 15), the corresponding acetylated lesions seem to adopt the syn-conformation with high preference (10,16,17). Crystal structures of Ellenberger, Beese, and Patel, showing the nonacetylated AF-dG-lesion inside different polymerases, prove that the lesion is indeed bound in anti conformation, allowing Watson-Crick base pairing with an incoming dCTP (18)(19)(20). While bypass of the nonacetylated lesions by high-fidelity polymerases is rather well understood, the mechanism that allows low-fidelity polymerases such as Pol η to replicate...

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“…114,115,117,119,120 This mutagenic chemical is believed to cause cell damage in humans by attacking and binding to guanine in DNA after being metabolized into bio-activated metabolites, N-hydroxy-PhIP and N-acetoxyl-PhIP by various cytochrome P450 enzymes, CYP1A1, 1A2, 1B1, N-acetyl transferases (NAT) and sulfotransferases (ST) in the liver. [121][122][123][124][125] Although many reports indicated that N-OH-PhIP is responsible for binding to DNA, 105,108,109 it is thought that this compound requires further activation by the action of NAT and ST before attacking. 120,126,127 Indeed, reports have recently found arylamine-DNA adducts (dG-C8-PhIP) in human tissues.…”

Section: Purification Of Bilirubinmentioning

confidence: 99%

“…The metabolism of mutagens such as 1.7 (BP) and 1.8 (PhIP) 5,70,[98][99][100]105,111,138,139 by CYP enzymes have been well studied in relation to the mechanism of their mutagenicity. 70,72,98,140 All of the environmental mutagens studied are in fact precursor molecules to the actual carcinogenic compounds which are formed by the action of CYP enzymes.…”

Section: Purification Of Bilirubinmentioning

confidence: 99%

“…88,90 Therefore, Bhosale et al 88 suggested that experiments involving in 1.3 (PRO) should be performed in the dark to avoid the formation of photooxidation products. [105][106][107] It is important to note that 1.5 (2AF) is oxidized by cytochrome P450 enzymes to the more active electrophilic agent, N-hydroxy-2-aminofluorene (N-OH-2-AF) and then HOSO 2 O-NH-2-AF under the action of sulfotransferase or N-acyltransferase. They are both considered the most likely to attack the nucleophilic positions in DNA such as C8-arylamine-dG, C8-arylamine-dA, and the amino group of deoxyguaninosine as depicted in Figure 1 The level of 1.8 (PhIP) in food varies greatly depending on type of food and cooking method.…”

Section: Purification Of Bilirubinmentioning

confidence: 99%

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Physical and chemical interactions between bile pigments and polyaromatic mutagens

Trieu¹

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A major cause of cancer in humans is exposure to mutagenic compounds. This raises the question of how humans can be protected from these environmental mutagens. Bile pigments (BPs) such as biliverdin, unconjugated bilirubin and protoporphyrin and their derivatives have recently been found to act as antioxidants and inhibit the mutagenic effects of several known environmental mutagens including 2-aminofluorene, benzo [α]pyrene, and 2-amino-1-methyl-6-phenylimido [4,5-b]pyridine. Despite these promising results, very little is known about the mechanisms by which this inhibition is achieved. Understanding these mechanisms would be useful for future drug development. Therefore, this PhD thesis aims to explore physical and chemical interactions between BPs and mutagens. Effects of BPs on the bioavailability and metabolism of mutagens were also examined in vitro using the colorectal adenocarcinoma (Caco-2 cell) monolayer model and the human liver S9 fraction.The physical interactions between mutagens and BPs were examined using three different methods:NMR, UV and effects of bioavailability. The results of the comparison of the NMR spectra of mutagens in the absence and presence of BPs showed very little changes in the chemical shifts of the protons and the changes that did occur were the result of acid/base interactions between the BPs and mutagens. The UV spectrum of each mutagen was measured in the presence and absence of varying concentrations of BPs, and there were no changes to the UV spectra of any of the compounds. Strong physical interactions or aggregation of compounds can also affect their absorption across cell monolayers and so the apparent permeability of mutagens across Caco-2 cell monolayers in the presence and absence of BPs were measured. The results indicated that BPs increased the permeability of the mutagens slightly and effected how much of the compounds remained in tight association with the monolayer but the effects were small. These experiments provided evidence to suggest that physical interactions and aggregations are unlikely to be a major contributing mechanism of the inhibitory effects of BPs on environmental mutagens.Chemical reactions between BPs and the DNA modifying metabolites of mutagens (epoxides) were studied using styrene epoxide as a model for the reactive metabolites. Styrene epoxide is commercially available, stable and less toxic than the reactive metabolites of the mutagens.Competitive reactions were performed in which BPs and their derivatives were placed in solution with guanine and allowed to react with styrene epoxide. These reactions showed that BPs and their dimethyl esters are more reactive to the epoxide than guanine. Bile pigments primarily react through their carboxylic acid groups with the mono-and di-styrene epoxide esters being the major products isolated form the reactions. The pyrrole rings in bilirubin also showed some evidence of iii reaction with styrene epoxide though the products were too unstable to isolate. Thus, it was clear that BPs can effecti...

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Mechanism of error-free and semitargeted mutagenic bypass of an aromatic amine lesion by Y-family polymerase Dpo4

Cited by 45 publications

References 59 publications

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

Mechanism of replication blocking and bypass of Y-family polymerase η by bulky acetylaminofluorene DNA adducts

Physical and chemical interactions between bile pigments and polyaromatic mutagens

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