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Structural and functional analysis of Sulfolobus solfataricus Y-family DNA polymerase Dpo4-catalyzed bypass of the malondialdehyde-deoxyguanosine adduct.

ABSTRACT: Oxidative stress can induce the formation of reactive electrophiles, such as DNA peroxidation products, e.g., base propenals, and lipid peroxidation products, e.g., malondialdehyde. Base propenals and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG). When paired opposite cytosine in duplex DNA at physiological pH, M1dG undergoes ring opening to form N2-(3-oxo-1-propenyl)-dG (N2-OPdG). Previous work has shown that M1dG is mutagenic in bacteria and mammalian cells and that its mutagenicity in Escherichia coli is dependent on induction of the SOS response, indicating a role for translesion DNA polymerases in the bypass of M1dG. To probe the mechanism by which translesion polymerases bypass M1dG, kinetic and structural studies were conducted with a model Y-family DNA polymerase, Dpo4 from Sulfolobus solfataricus. The level of steady-state incorporation of dNTPs opposite M1dG was reduced 260-2900-fold and exhibited a preference for dATP incorporation. Liquid chromatography-tandem mass spectrometry analysis of the full-length extension products revealed a spectrum of products arising principally by incorporation of dC or dA opposite M1dG followed by partial or full-length extension. A greater proportion of -1 deletions were observed when dT was positioned 5' of M1dG. Two crystal structures were determined, including a "type II" frameshift deletion complex and another complex with Dpo4 bound to a dC.M1dG pair located in the postinsertion context. Importantly, M1dG was in the ring-closed state in both structures, and in the structure with dC opposite M1dG, the dC residue moved out of the Dpo4 active site, into the minor groove. The results are consistent with the reported mutagenicity of M1dG and illustrate how the lesion may affect replication events.

SUBMITTER: Eoff RL

PROVIDER: S-EPMC2717710 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Structural and functional analysis of Sulfolobus solfataricus Y-family DNA polymerase Dpo4-catalyzed bypass of the malondialdehyde-deoxyguanosine adduct.

Eoff Robert L RL Stafford Jennifer B JB Szekely Jozsef J Rizzo Carmelo J CJ Egli Martin M Guengerich F Peter FP Marnett Lawrence J LJ

Biochemistry 20090801 30

Oxidative stress can induce the formation of reactive electrophiles, such as DNA peroxidation products, e.g., base propenals, and lipid peroxidation products, e.g., malondialdehyde. Base propenals and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG). When paired opposite cytosine in duplex DNA at physiological pH, M1dG undergoes ring opening to form N2-(3-oxo-1-propenyl)-dG (N2-OPdG). Previous work has ...[more]

PMID: 19492857

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Project description:trans-4-Hydroxynonenal (HNE) is the major peroxidation product of ?-6 polyunsaturated fatty acids in vivo. Michael addition of the N(2)-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N(2)-dGuo (1,N(2)-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N(2)-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N(2)-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua ? Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, the (6S,8R,11S)-1,N(2)-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either dGTP or dATP, was positioned with Watson-Crick pairing opposite the template 5'-neighbor base, dCyt or dThy, respectively. In contrast, for the 18-mer:14-mer template-primers with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, ring opening of the adduct to the corresponding N(2)-dGuo aldehyde species occurred. This allowed Watson-Crick base pairing at the (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair.

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Dataset Information

Structural and functional analysis of Sulfolobus solfataricus Y-family DNA polymerase Dpo4-catalyzed bypass of the malondialdehyde-deoxyguanosine adduct.

Publications

Structural and functional analysis of Sulfolobus solfataricus Y-family DNA polymerase Dpo4-catalyzed bypass of the malondialdehyde-deoxyguanosine adduct.

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