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The Role of Mismatch Repair, Base Excision Repair and PARP-1 in the Processing of Oxidative DNA Damage


Repmann, Simone. The Role of Mismatch Repair, Base Excision Repair and PARP-1 in the Processing of Oxidative DNA Damage. 2014, University of Zurich, Faculty of Science.

Abstract

Our DNA is constantly exposed to spontaneous oxidative stress, which results from metabolic byproducts and which represents a key threat to genomic stability. Oxidation of DNA results in accumulation of the mutagenic 8-hydroxy-2’- deoxyguanosine (GO), which is mainly removed by the base excision repair (BER) pathway. G/C base pairs are oxidized to GO/Cs, which are recognized by oxoguanine DNA glycosylase (OGG1) and corrected to G/Cs, with the assistance of the downstream BER machinery. During replication, GO can mispair with adenine. GO/A mispairs are addressed by MYH-initiated BER that removes the As and replaces them with Cs to generate GO/Cs, which are substrates for OGG1- dependent BER. Dysfunction of MYH leads to G:C to T:A transversions and is linked to MYH-associated polyposis (MAP).
During the past decade, repair of oxidative DNA lesions such as GO was shown to involve also the postreplicative mismatch repair system (MMR), but its role within this process remained obscure. GO/A mispairs were known to be poor substrates for MMR, but we show here that, in an in vitro MMR assay based on circular lesion- containing plasmids and human cell extracts, the intermediates of GO/A - but not GO/C - processing by BER can serve as strand discrimination signals for MMR. These observations were not restricted to human cell extracts, but were observed also in extracts of Xenopus laevis eggs. During S-phase, GO would be present predominantly in the parental DNA strand. Thus, “hijacking” of strand breaks arising during MYH-dependent GO/A processing by BER would help direct the mismatch repair process correctly to the daughter strand. In contrast, OGG1- generated nicks at GO/C sites would direct MMR erroneously to the parental strand. Our data thus show that BER-mediated processing of oxidative DNA damage is coordinated to help MMR improve replication fidelity.
In a complementary study, we set out to study the interactome of the mismatch recognition factor MutSα during oxidative DNA damage response. We had hoped that identification of interaction partners of these MMR proteins might help us understand the details of the interplay of the different pathways of DNA metabolism and DNA damage signaling in cells subjected to oxidative stress. I generated a novel set of expression vectors and cell lines that could be used for future mass spectrometric analysis of the interacting partners of MutSα. I also confirmed that MMR deficiency leads to a decreased cell survival and a prolonged G2/M arrest during oxidative stress.
Poly(ADP-ribose) polymerase 1(PARP-1) is an enzyme that is extremely rapidly activated by single-strand breaks (SSBs), to which it binds with high affinity. This 'nick protector' enzyme has been assigned a role in BER that is currently ill- defined, however, it has recently acquired substantial clinical importance, because its inhibition effectively kills BRCA1/-2 deficient breast and ovarian cancer cells.
PARP inhibition is generally believed to give rise to toxic SSB/PARP-1 complexes that can only be repaired in the presence of BRCA1/-2, but the underlying causes of those SSBs are undefined yet. We wanted to learn whether these breaks might stem from the processing of spontaneous oxidative damage. Using cell survival assays with human BRCA1-deficient cell lines, we show that MYH knockdown attenuates the toxic effect of PARP inhibitors. These findings show that DNA oxidation is a source of lesions that contribute to the toxicity of PARP inhibition in BRCA-deficient cells and suggest that augmentation of oxidative damage processing might increase the efficacy of PARP inhibitors in the clinic.

Abstract

Our DNA is constantly exposed to spontaneous oxidative stress, which results from metabolic byproducts and which represents a key threat to genomic stability. Oxidation of DNA results in accumulation of the mutagenic 8-hydroxy-2’- deoxyguanosine (GO), which is mainly removed by the base excision repair (BER) pathway. G/C base pairs are oxidized to GO/Cs, which are recognized by oxoguanine DNA glycosylase (OGG1) and corrected to G/Cs, with the assistance of the downstream BER machinery. During replication, GO can mispair with adenine. GO/A mispairs are addressed by MYH-initiated BER that removes the As and replaces them with Cs to generate GO/Cs, which are substrates for OGG1- dependent BER. Dysfunction of MYH leads to G:C to T:A transversions and is linked to MYH-associated polyposis (MAP).
During the past decade, repair of oxidative DNA lesions such as GO was shown to involve also the postreplicative mismatch repair system (MMR), but its role within this process remained obscure. GO/A mispairs were known to be poor substrates for MMR, but we show here that, in an in vitro MMR assay based on circular lesion- containing plasmids and human cell extracts, the intermediates of GO/A - but not GO/C - processing by BER can serve as strand discrimination signals for MMR. These observations were not restricted to human cell extracts, but were observed also in extracts of Xenopus laevis eggs. During S-phase, GO would be present predominantly in the parental DNA strand. Thus, “hijacking” of strand breaks arising during MYH-dependent GO/A processing by BER would help direct the mismatch repair process correctly to the daughter strand. In contrast, OGG1- generated nicks at GO/C sites would direct MMR erroneously to the parental strand. Our data thus show that BER-mediated processing of oxidative DNA damage is coordinated to help MMR improve replication fidelity.
In a complementary study, we set out to study the interactome of the mismatch recognition factor MutSα during oxidative DNA damage response. We had hoped that identification of interaction partners of these MMR proteins might help us understand the details of the interplay of the different pathways of DNA metabolism and DNA damage signaling in cells subjected to oxidative stress. I generated a novel set of expression vectors and cell lines that could be used for future mass spectrometric analysis of the interacting partners of MutSα. I also confirmed that MMR deficiency leads to a decreased cell survival and a prolonged G2/M arrest during oxidative stress.
Poly(ADP-ribose) polymerase 1(PARP-1) is an enzyme that is extremely rapidly activated by single-strand breaks (SSBs), to which it binds with high affinity. This 'nick protector' enzyme has been assigned a role in BER that is currently ill- defined, however, it has recently acquired substantial clinical importance, because its inhibition effectively kills BRCA1/-2 deficient breast and ovarian cancer cells.
PARP inhibition is generally believed to give rise to toxic SSB/PARP-1 complexes that can only be repaired in the presence of BRCA1/-2, but the underlying causes of those SSBs are undefined yet. We wanted to learn whether these breaks might stem from the processing of spontaneous oxidative damage. Using cell survival assays with human BRCA1-deficient cell lines, we show that MYH knockdown attenuates the toxic effect of PARP inhibitors. These findings show that DNA oxidation is a source of lesions that contribute to the toxicity of PARP inhibition in BRCA-deficient cells and suggest that augmentation of oxidative damage processing might increase the efficacy of PARP inhibitors in the clinic.

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Additional indexing

Item Type:Dissertation
Referees:Jiricny J, Aebersold Ruedi, Bignami Margherita
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2014
Deposited On:20 Jan 2015 15:04
Last Modified:23 May 2016 16:27

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