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Pre-Replicative Repair of Oxidized Bases Maintains Fidelity in Mammalian Genomes: The Cowcatcher Role of NEIL1 DNA Glycosylase.

ABSTRACT: Genomic fidelity in the humans is continuously challenged by genotoxic reactive oxygen species (ROS) generated both endogenously during metabolic processes, and by exogenous agents. Mispairing of most ROS-induced oxidized base lesions during DNA replication induces mutations. Although bulky base adducts induced by ultraviolet light and other environmental mutagens block replicative DNA polymerases, most oxidized base lesions do not block DNA synthesis. In 8-oxo-G:A mispairs generated by the incorporation of A opposite unrepaired 8-oxo-G, A is removed by MutYH (MYH) for post-replicative repair, and other oxidized base lesions must be repaired prior to replication in order to prevent mutation fixation. Our earlier studies documented S phase-specific overexpression of endonuclease VIII-like 1 (NEIL1) DNA glycosylase (DG), one of five oxidized base excision repair (BER)-initiating enzymes in mammalian cells, and its high affinity for replication fork-mimicking single-stranded (ss)DNA substrates. We recently provided experimental evidence for the role of NEIL1 in replicating-strand repair, and proposed the "cowcatcher" model of pre-replicative BER, where NEIL1's nonproductive binding to the lesion base in ssDNA template blocks DNA chain elongation, causing fork regression. Repair of the lesion in the then re-annealed duplex is carried out by NEIL1 in association with the DNA replication proteins. In this commentary, we highlight the critical role of pre-replicative BER in preventing mutagenesis, and discuss the distinction between pre-replicative vs. post-replicative BER.

SUBMITTER: Rangaswamy S

PROVIDER: S-EPMC5541308 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Pre-Replicative Repair of Oxidized Bases Maintains Fidelity in Mammalian Genomes: The Cowcatcher Role of NEIL1 DNA Glycosylase.

Rangaswamy Suganya S Pandey Arvind A Mitra Sankar S Hegde Muralidhar L ML

Genes 20170630 7

Genomic fidelity in the humans is continuously challenged by genotoxic reactive oxygen species (ROS) generated both endogenously during metabolic processes, and by exogenous agents. Mispairing of most ROS-induced oxidized base lesions during DNA replication induces mutations. Although bulky base adducts induced by ultraviolet light and other environmental mutagens block replicative DNA polymerases, most oxidized base lesions do not block DNA synthesis. In 8-oxo-G:A mispairs generated by the inco ...[more]

PMID: 28665322

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Project description:The DNA glycosylase hNEIL1 initiates the base excision repair (BER) of a diverse array of lesions, including ring-opened purines and saturated pyrimidines. Of these, the hydantoin lesions, guanidinohydantoin (Gh) and the two diastereomers of spiroiminodihydantoin (Sp1 and Sp2), have garnered much recent attention due to their unusual structures, high mutagenic potential, and detection in cells. In order to provide insight into the role of repair, the excision efficiency by hNEIL1 of these hydantoin lesions relative to other known substrates was determined. Most notably, quantitative examination of the substrate specificity with hNEIL1 revealed that the hydantoin lesions are excised much more efficiently (>100-fold faster) than the reported standard substrates thymine glycol (Tg) and 5-hydroxycytosine (5-OHC). Importantly, the glycosylase and beta,delta-lyase reactions are tightly coupled such that the rate of the lyase activity does not influence the observed substrate specificity. The activity of hNEIL1 is also influenced by the base pair partner of the lesion, with both Gh and Sp removal being more efficient when paired with T, G, or C than when paired with A. Notably, the most efficient removal is observed with the Gh or Sp paired in the unlikely physiological context with T; indeed, this may be a consequence of the unstable nature of base pairs with T. However, the facile removal via BER in promutagenic base pairs that are reasonably formed after replication (such as Gh.G) may be a factor that modulates the mutagenic profile of these lesions. In addition, hNEIL1 excises Sp1 faster than Sp2, indicating the enzyme can discriminate between the two diastereomers. This is the first time that a BER glycosylase has been shown to be able to preferentially excise one diastereomer of Sp. This may be a consequence of the architecture of the active site of hNEIL1 and the structural uniqueness of the Sp lesion. These results indicate that the hydantoin lesions are the best substrates identified thus far for hNEIL1 and suggest that repair of these lesions may be a critical function of the hNEIL1 enzyme in vivo.

Project description:Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ?30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ?3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, ?-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all the measured DNA lesions, imidazole ring-opened 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were preferentially released by both NEIL1 enzymes with K242R being ?1.3 and 1.2-fold more efficient than K242 on excision of FapyAde and FapyGua, respectively. Consistent with the prior literature, large differences (?7.5 to 12-fold) were measured in the excision of ThyGly from genomic DNA by the unedited versus edited NEIL1. In contrast, the edited NEIL1 was more efficient (?3 to 5-fold) on release of 5-hydroxycytosine. Excision kinetics on DNA containing a site-specific aflatoxin B1-FapyGua adduct revealed an ?1.4-fold higher rate by the unedited NEIL1. Molecular modeling provides insight into these differential substrate specificities. The results of this study and in particular, the comparison of substrate specificities of unedited and edited NEIL1 using biologically and clinically important base lesions, are critical for defining its role in preservation of genomic integrity.

Dataset Information

Pre-Replicative Repair of Oxidized Bases Maintains Fidelity in Mammalian Genomes: The Cowcatcher Role of NEIL1 DNA Glycosylase.

Publications

Pre-Replicative Repair of Oxidized Bases Maintains Fidelity in Mammalian Genomes: The Cowcatcher Role of NEIL1 DNA Glycosylase.

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