Compromised Incision of Oxidized Pyrimidines in Liver Mitochondria of Mice Deficient in NTH1 and OGG1 Glycosylases

Abstract

Mitochondrial DNA is constantly exposed to high levels of endogenously produced reactive oxygen species, resulting in elevated levels of oxidative damaged DNA bases. A large spectrum of DNA base alterations can be detected after oxidative stress, and many of these are highly mutagenic. Thus, an efficient repair of these is necessary for survival. Some of the DNA repair pathways involved have been characterized, but others are not yet determined. A DNA repair activity for thymine glycol and other oxidized pyrimidines has been described in mammalian mitochondria, but the nature of the glycosylases involved in this pathway remains unclear. The generation of mouse strains lacking murine thymine glycol-DNA glycosylase (mNTH1) and/or murine 8-oxoguanine-DNA glycosylase (mOGG1), the two major DNA N-glycosylase/apurinic/apyrimidinic (AP) lyases involved in the repair of oxidative base damage in the nucleus, has provided very useful biological model systems for the study of the function of these and other glycosylases in mitochondrial DNA repair. In this study, mouse liver mitochondrial extracts were generated from mNTH1-, mOGG1-, and [mNTH1, mOGG1]-deficient mice to ascertain the role of each of these glycosylases in the repair of oxidized pyrimidine base damage. We also characterized for the first time the incision of various modified bases in mitochondrial extracts from a doubleknock-out [mNTH1, mOGG1]-deficient mouse. We show that mNTH1 is responsible for the repair of thymine glycols in mitochondrial DNA, whereas other glycosylase/AP lyases also participate in removing other oxidized pyrimidines, such as 5-hydroxycytosine and 5-hydroxyuracil. We did not detect a backup glycosylase or glycosylase/AP lyase activity for thymine glycol in the mitochondrial mouse extracts.Under normal metabolic conditions, reactive oxygen species are generated primarily as a by-product of the electron transport chain in mitochondria (1). The mtDNA represents a particularly susceptible target for reactive oxygen species, as it is closely associated with the mitochondrial inner membrane where most reactive oxygen species are generated (2). Various laboratories (3, 4), including ours (5), have demonstrated that oxidative DNA base damage accumulates more in the mitochondrial DNA than in the nuclear DNA.The endonuclease III (NTH) 1 family of glycosylases is a highly conserved class of repair enzymes that are found from bacteria to man (6). NTH1 is the main endonuclease III homologue found in mice and humans (7-9). Its glycosylase/AP lyase component has very broad substrate specificity, and it removes several different types of oxidized bases from DNA (10, 11). The primary target for NTH1 is oxidized pyrimidines in DNA, and NTH1 is involved in the repair of thymine glycol (5,6-dihydroxy-5,6-dihydrothymine (Tg)) in vivo (12).Our laboratory has previously demonstrated the existence of a repair system specific for thymine glycols in mammalian mitochondria (13). A Tg glycosylase/AP lyase (mtTGEndo) was isolated from rat liver mitochondria to near-...

Regulation of reactive oxygen species, DNA damage and c-Myc function by peroxiredoxin 1

“…Other cyclopurine-2 0 -deoxynucleosides such as (5 0 R)-cdA, (5 0 R)-cdG, and (5 0 S)-cdG are also likely to be processed by NE because of a covalent bond between the sugar and base moieties of the same nucleoside, thus excluding repair by BE (Dizdaroglu, 1986). However, we have found equivalent activities of uracil DNA glycosylase, oxoguanine DNA glycosylase, and endonuclease III homologue I in prdx1 þ / þ and prdx1À/À liver and kidney extracts (data not shown) (de Souza-Pinto et al, 2001;Karahalil et al, 2003). These findings therefore do not account for the higher levels of DNA damage in prdx1À/À mice.…”

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…At the same time, as we have already pointed out, the glycosylases do have preferred substrates and it is also likely that the individual enzyme acts preferentially depending on the state of the genome, i.e., whether the cells are cycling or postmitotic and whether the damage is located in transcriptionally active vs. inactive sequences [68]. This could also explain why accumulation of oxidized bases in the genomes of OGG1 or NTH1 null mouse cells does not cause any obvious phenotype [69]. Although oxidized bases could possibly be repaired alternatively via the NER pathway, this is unlikely to be a major alternative repair mode for most oxidized bases [70], because such bases do not induce significant perturbations in the duplex DNA structure needed for recognition by the NER proteins.…”

“…Lig IIIb is a splice variant of the nuclear Lig IIIa, whereas Polg is unique for the mitochondria [156]. Similarly, splice variants of nuclear OGG1 and NTH1 have been characterized in mitochondria [69,157]. Mitochondrial transport of proteins generally requires an N-terminal mitochondrial targeting sequence (MTS) which is cleaved off in the mitochondrial matrix by specific peptidases [158] although, some mitochondrial proteins lacking MTS, utilize an internal targeting sequence…”

p53 functions in the incorporation step in DNA base excision repair in mouse liver mitochondria

“…Each DNA glycosylase recognizes a defined set of modified bases, and thus these enzymes provide the specificity of the repair process . In mitochondria, three major DNA glycosylases have been characterized: mtUDG, which catalyses the removal of uracil (Domena and Mosbaugh, 1985); mtOGG1, which removes oxidized purines, most notably 8-oxoG (SouzaPinto et al, 2001); and mtNTH1, which recognizes oxidized pyrimidines (Karahalil et al, 2003). We measured the activities of each of these enzymes in mitochondrial and nuclear extracts from wt, p53 À/ þ and p53 À/À mice to investigate whether p53 levels modulate activity of those enzymes.…”