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 double-knock-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.
Highlights
Mouse liver mitochondrial extracts were generated there is good evidence for the presence of an endofrom mNTH1, mOGG1, and [mNTH1, mOGG1]-deficient nuclease III homologue in Saccharomyces cerevisiae mitochonmice to ascertain the role of each of these glycosylases in dria (Ntg1p) [14], the situation in mammalian mitochondria is the repair of oxidized pyrimidine base damage
We show that mNTH1 is responsible for the repair of thymine glycols in mitochondrial DNA, whereas other glycosylase/AP lyases participate in removing other oxidized pyrimidines, such as 5-hydroxycytosine and 5-hydroxyuracil
Both the single knock-outs and the double-knock-out extracts contained higher AP-endonuclease activity than the wild type (Fig. 2D), suggesting a possible compensatory mechanism, because mOGG1 and mNTH1 are bifunctional glycosylases with associated AP lyase activities. These results suggest that the absence of mNTH1 or mOGG1 did not interfere with BER of lesions that are recognized by another glycosylase, such as UDG
Summary
Vol 278, No 36, Issue of September 5, pp. 33701–33707, 2003 Printed in U.S.A. Compromised Incision of Oxidized Pyrimidines in Liver Mitochondria of Mice Deficient in NTH1 and OGG1 Glycosylases*. Mouse liver mitochondrial extracts were generated there is good evidence for the presence of an endofrom mNTH1-, mOGG1-, and [mNTH1, mOGG1]-deficient nuclease III homologue in Saccharomyces cerevisiae mitochonmice to ascertain the role of each of these glycosylases in dria (Ntg1p) [14], the situation in mammalian mitochondria is the repair of oxidized pyrimidine base damage. We show that mNTH1 is responsible for the repair of thymine glycols in mitochondrial DNA, whereas other glycosylase/AP lyases participate in removing other oxidized pyrimidines, such as 5-hydroxycytosine and 5-hydroxyuracil. This is the first characterization of BER enzymes in mitochondria from doubleknock-out mice deficient in the two major DNA glycosylases for the repair of oxidized bases
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