scholarly journals The Effect ofMsh2Knockdown on Toxicity Induced bytert-Butyl-hydroperoxide, Potassium Bromate, and Hydrogen Peroxide in Base Excision Repair Proficient and Deficient Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
N. Cooley ◽  
R. H. Elder ◽  
A. C. Povey

The DNA mismatch repair (MMR) and base excision repair (BER) systems are important determinants of cellular toxicity following exposure to agents that cause oxidative DNA damage. To examine the interactions between these different repair systems, we examined whether toxicity, induced byt-BOOH and KBrO3, differs in BER proficient (Mpg+/+,Nth1+/+) and deficient (Mpg−/−,Nth1−/−) mouse embryonic fibroblasts (MEFs) followingMsh2knockdown of between 79 and 88% using an shRNA expression vector.Msh2knockdown inNth1+/+cells had no effect ont-BOOH and KBrO3induced toxicity as assessed by an MTT assay; knockdown inNth1−/−cells resulted in increased resistance tot-BOOH and KBrO3, a result consistent with Nth1 removing oxidised pyrimidines.Msh2knockdown inMpg+/+cells had no effect ont-BOOH toxicity but increased resistance to KBrO3; inMpg−/−cells,Msh2knockdown increased cellular sensitivity to KBrO3but increased resistance to t-BOOH, suggesting a role forMpgin removing DNA damage induced by these agents. MSH2 dependent and independent pathways then determine cellular toxicity induced by oxidising agents. A complex interaction between MMR and BER repair systems, that is, exposure dependent, also exists to determine cellular toxicity.

2008 ◽  
Vol 29 (3) ◽  
pp. 794-807 ◽  
Author(s):  
Lyra M. Griffiths ◽  
Dan Swartzlander ◽  
Kellen L. Meadows ◽  
Keith D. Wilkinson ◽  
Anita H. Corbett ◽  
...  

ABSTRACT DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.


2008 ◽  
Vol 30 (1) ◽  
pp. 2-10 ◽  
Author(s):  
S. Maynard ◽  
S. H. Schurman ◽  
C. Harboe ◽  
N. C. de Souza-Pinto ◽  
V. A. Bohr

DNA Repair ◽  
2005 ◽  
Vol 4 (11) ◽  
pp. 1270-1280 ◽  
Author(s):  
Takanori Sugimoto ◽  
Emi Igawa ◽  
Haruna Tanihigashi ◽  
Mayumi Matsubara ◽  
Hiroshi Ide ◽  
...  

2001 ◽  
Vol 38 (2-3) ◽  
pp. 180-190 ◽  
Author(s):  
Sankar Mitra ◽  
Istvan Boldogh ◽  
Tadahide Izumi ◽  
Tapas K. Hazra

Toxicology ◽  
2003 ◽  
Vol 193 (1-2) ◽  
pp. 43-65 ◽  
Author(s):  
Tadahide Izumi ◽  
Lee R. Wiederhold ◽  
Gargi Roy ◽  
Rabindra Roy ◽  
Arun Jaiswal ◽  
...  

2008 ◽  
Vol 28 (16) ◽  
pp. 4975-4987 ◽  
Author(s):  
Pingfang Liu ◽  
Limin Qian ◽  
Jung-Suk Sung ◽  
Nadja C. de Souza-Pinto ◽  
Li Zheng ◽  
...  

ABSTRACT Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision repair (SP-BER) replacing a single nucleotide. However, certain oxidative lesions cannot be processed by SP-BER. Here we report that 2-deoxyribonolactone (dL), a major type of oxidized abasic site, inhibits replication by mitochondrial DNA (mtDNA) polymerase γ and interferes with SP-BER by covalently trapping polymerase γ during attempted dL excision. However, repair of dL was detected in human mitochondrial extracts, and we show that this repair is via long-patch BER (LP-BER) dependent on flap endonuclease 1 (FEN1), not previously known to be present in mitochondria. FEN1 was retained in protease-treated mitochondria and detected in mitochondrial nucleoids that contain known mitochondrial replication and transcription proteins. Results of immunofluorescence and subcellular fractionation studies were also consistent with the presence of FEN1 in the mitochondria of intact cells. Immunodepletion experiments showed that the LP-BER activity of mitochondrial extracts was strongly diminished in parallel with the removal of FEN1, although some activity remained, suggesting the presence of an additional flap-removing enzyme. Biological evidence for a FEN1 role in repairing mitochondrial oxidative DNA damage was provided by RNA interference experiments, with the extent of damage greater and the recovery slower in FEN1-depleted cells than in control cells. The mitochondrial LP-BER pathway likely plays important roles in repairing dL lesions and other oxidative lesions and perhaps in normal mtDNA replication.


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