Dataset Information

Translesion DNA synthesis by human DNA polymerase eta on templates containing a pyrimidopurinone deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one.

ABSTRACT: M(1)dG (3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one) lesions are mutagenic in bacterial and mammalian cells, leading to base substitutions (mostly M(1)dG to dT and M(1)dG to dA) and frameshift mutations. M(1)dG is produced endogenously through the reaction of peroxidation products, base propenal or malondialdehyde, with deoxyguanosine residues in DNA. The mutagenicity of M(1)dG in Escherichia coli is dependent on the SOS response, specifically the umuC and umuD gene products, suggesting that mutagenic lesion bypass occurs by the action of translesion DNA polymerases, like DNA polymerase V. Bypass of DNA lesions by translesion DNA polymerases is conserved in bacteria, yeast, and mammalian cells. The ability of recombinant human DNA polymerase eta to synthesize DNA across from M(1)dG was studied. M(1)dG partially blocked DNA synthesis by polymerase eta. Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M(1)dG lesion (800-fold less efficient than opposite dG). Extension from M(1)dG.dC was equally as efficient as from control primer-templates (dG.dC). dATP insertion opposite M(1)dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M(1)dG.dA was 20-fold less efficient than dG.dC. The sequences of full-length human DNA polymerase eta bypass products of M(1)dG were determined by LC-ESI/MS/MS. Bypass products contained incorporation of dA (52%) or dC (16%) opposite M(1)dG or -1 frameshifts at the lesion site (31%). Human DNA polymerase eta bypass may lead to M(1)dG to dT and frameshift but likely not M(1)dG to dA mutations during DNA replication.

SUBMITTER: Stafford JB

PROVIDER: S-EPMC2651650 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Translesion DNA synthesis by human DNA polymerase eta on templates containing a pyrimidopurinone deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one.

Stafford Jennifer B JB Eoff Robert L RL Kozekova Albena A Rizzo Carmelo J CJ Guengerich F Peter FP Marnett Lawrence J LJ

Biochemistry 20090101 2

M(1)dG (3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one) lesions are mutagenic in bacterial and mammalian cells, leading to base substitutions (mostly M(1)dG to dT and M(1)dG to dA) and frameshift mutations. M(1)dG is produced endogenously through the reaction of peroxidation products, base propenal or malondialdehyde, with deoxyguanosine residues in DNA. The mutagenicity of M(1)dG in Escherichia coli is dependent on the SOS response, specifically the umuC and umuD gene ...[more]

PMID: 19108641

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Project description:Translesion synthesis (TLS) of the N(2)-2'-deoxyguanosine (dG-N(2)-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5'-CG1G2CG3CC-3'). TLS efficiency was 38%, 29%, and 25% for the dG-N(2)-IQ located at G1, G2, and G3, respectively, which suggests that dG-N(2)-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8-35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N(2)-IQ bypass. Mutation frequencies (MFs) of dG-N(2)-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ( ( 2015 ) Nucleic Acids Res. 43 , 8340 - 8351 ). The major type of mutation induced by dG-N(2)-IQ was targeted G → T, as was reported for dG-C8-IQ. In each site, knockdown of pol κ resulted in an increase in MF, whereas MF was reduced when pol η, pol ι, pol ζ, or Rev1 was knocked down. The reduction in MF was most pronounced when pol η, pol ζ, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol κ predominantly performs error-free TLS of the dG-N(2)-IQ adduct, whereas pols η, pol ζ, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κ showed that the former was inefficient in full-length primer extension on dG-N(2)-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular results may arise from several factors including the crucial roles played by the accessory proteins in TLS.

Dataset Information

Translesion DNA synthesis by human DNA polymerase eta on templates containing a pyrimidopurinone deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one.

Publications

Translesion DNA synthesis by human DNA polymerase eta on templates containing a pyrimidopurinone deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one.

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