Characterization of Two Independent Amino Acid Substitutions that Disrupt the DNA Repair Functions of the Yeast Apn1

Jilani, Arshad; Vongsamphanh, Ratsavarinh; Leduc, Anick; Gros, Laurent; Saparbaev, Murat; Ramotar, Dindial

doi:10.1021/bi034163m

Cited by 24 publications

(19 citation statements)

References 30 publications

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“…Purification of GST and His-tagged Proteins-GST-GAPDH fusion proteins were overexpressed in E. coli BW528 strain and purified by glutathione-Sepharose 4B mini columns as previously described (23), and the His 6 -APE1 was purified using Talon affinity beads according to the manufacturer's protocol (Clontech).…”

Section: Methodsmentioning

confidence: 99%

Human Glyceraldehyde-3-phosphate Dehydrogenase Plays a Direct Role in Reactivating Oxidized Forms of the DNA Repair Enzyme APE1

Azam¹,

Jouvet

Jilani

et al. 2008

Journal of Biological Chemistry

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121

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has diverse biological functions including its nuclear translocation in response to oxidative stress. We show that GAPDH physically associates with APE1, an essential enzyme involved in the repair of abasic sites in damaged DNA, as well as in the redox regulation of several transcription factors. This interaction allows GAPDH to convert the oxidized species of APE1 to the reduced form, thereby reactivating its endonuclease activity to cleave abasic sites. The GAPDH variants C152G and C156G retain the ability to interact with but are unable to reactivate APE1, implicating these cysteines in catalyzing the reduction of APE1. Interestingly, GAPDH-small interfering RNA knockdown sensitized the cells to methyl methane sulfonate and bleomycin, which generate lesions that are repaired by APE1, but showed normal sensitivity to 254-nm UV. Moreover, the GAPDH knockdown cells exhibited an increased level of spontaneous abasic sites in the genomic DNA as a result of diminished APE1 endonuclease activity. Thus, the nuclear translocation of GAPDH during oxidative stress constitutes a protective mechanism to safeguard the genome by preventing structural inactivation of APE1.The evolutionary conserved enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 3 exists as a tetramer that catalyzes a critical reaction in the second stage of the glycolytic pathway (1). It uses the oxidized form of nicotinamide adenine dinucleotide (NAD ϩ ) and converts glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with the concomitant release of NADH in an oxido-reduction reaction (1). GAPDH is also a key redox-sensitive protein that possesses an active site cysteine sulfhydryl that is susceptible to oxidation (2). Under oxidative stress, GAPDH rapidly undergoes disulfide bond formation leading to reduction in its enzymatic activity (2, 3). GAPDH has the propensity to interact with several proteins that are vulnerable to aggregation and are associated with neurodegenerative disorders such as in the case of the pro-oxidant amyloid ␤ peptide involved in Alzheimer disease (4). Recent studies have documented that GAPDH is also involved in several other nuclear processes that include histone H2B gene expression, nuclear RNA export, apoptosis, and cellular response to DNA damage (5-8).Several lines of evidence support a role for GAPDH in DNA damage and repair (5, 9). For example, GAPDH can translocate from the cytoplasm to the nucleus when cells are challenged with the potent chemical oxidant and DNA-damaging agent H 2 O 2 , although it is not clear what is the function executed by GAPDH under this stress condition (10). However, a more recent study documented that nitric oxide can also induce nuclear localization of GAPDH where it is acetylated by the acetyltransferase p300/CBP via direct protein interaction, which in turn causes stimulation of the catalytic activity of p300/CBP, resulting in the activation of downstream targets such as p53 (11). Other studies have shown that GAPDH is associated w...

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Section: Methodsmentioning

confidence: 99%

Human Glyceraldehyde-3-phosphate Dehydrogenase Plays a Direct Role in Reactivating Oxidized Forms of the DNA Repair Enzyme APE1

Azam¹,

Jouvet

Jilani

et al. 2008

Journal of Biological Chemistry

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121

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“…Briefly, chromosomal DNA isolated from BLM-untreated and -treated (10 g/ml for 2 h) cells was assessed for the ability to permit incorporation of [methyl-3 H]dTMP by purified E. coli DNA polymerase 1 (Promega). Apn1 used for pretreating the chromosomal DNA was purified in this laboratory (30). Specific activity of the labeled [methyl-3 H]dTTP (NEN Life Sciences Products) was 1230 cpm/pmol.…”

Section: Methodsmentioning

confidence: 99%

“…genotoxicity measurement was determined by clonogenic assays of a parent strain YW465 and the DNA repair deficient mutant YW778 (Ref. 30; data not shown). For F-BLM uptake studies, exponentially growing cultures were washed twice in water and resuspended in 50 mM citrate acetate buffer (pH 5.5) containing 2% glucose and 0.05% Tween 20 at a density of 2 ϫ 10 8 cells/ml.…”

Section: Methodsmentioning

confidence: 99%

A Genome-Wide Screen in Saccharomyces cerevisiae Reveals Altered Transport As a Mechanism of Resistance to the Anticancer Drug Bleomycin

Pagé²,

et al. 2004

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The potent DNA damaging agent bleomycin (BLM) is highly effective for treating various cancers, although, in certain individuals, the development of cellular resistance to the drug can severely diminish its antineoplastic properties. We performed two independent genome-wide screens using a Saccharomyces cerevisiae mutant collection to isolate variants exhibiting either sensitivity or resistance to BLM. This procedure reproducibly identified a relatively large collection of 231 BLM-hypersensitive mutants, representing genes belonging to diverse functional groups. In contrast, only five BLM-resistant mutants could be recovered by our screens. Among these latter mutants, three were deleted for genes involved in plasma membrane transport, including the L-carnitine transporter Agp2, as well as the kinases Ptk2 and Sky1, which are involved in regulating polyamine transport. We further showed that Agp2 acts as a transporter of BLM and that overexpression of this transporter significantly enhances BLM-induced cell killing. Our data strongly implicate membrane transport as a key determinant in BLM resistance in yeast. This finding is critical, given that very little is known about BLM transport in human cells. Indeed, characterization of analogous mechanisms in humans may ultimately lead to enhancement of the antitumor properties of BLM.

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“…The pGst-MutT plasmid was isolated from yeast and amplified in bacteria, and the DNA sequence was verified. The vector pYES and pGst-MutT were introduced into the yeast strains, and MutT protein expression was verified by Western blotting with anti-GST antibodies as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

“…Later, using duplex oligonucleotides, Vance and Wilson (48) demonstrated that a glutathione S-transferase (GST)-tagged form of Apn1 can excise a single nucleotide at the 3Ј side of a nick, thereby generating a one-nucleotide gap. More recent studies revealed that Apn1 displayed the ability to remove more than one nucleotide following incision of an AP site (20). In fact, E. coli endonuclease IV (Nfo), a counterpart of Apn1, also possesses an intrinsic 3Ј35Ј exonuclease activity, which is very sensitive to ionic strength, metal ions, EDTA, and reducing conditions (25).…”

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confidence: 99%

The 3′→5′ Exonuclease of Apn1 Provides an Alternative Pathway To Repair 7,8-Dihydro-8-Oxodeoxyguanosine in Saccharomyces cerevisiae

Ищенко

Yang

Ramotar

et al. 2005

Molecular and Cellular Biology

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The 8-oxo-7,8-dihydrodeoxyguanosine (8oxoG), a major mutagenic DNA lesion, results either from direct oxidation of guanines or misincorporation of 8oxodGTP by DNA polymerases. At present, little is known about the mechanisms preventing the mutagenic action of 8oxodGTP in Saccharomyces cerevisiae. Herein, we report for the first time the identification of an alternative repair pathway for 8oxoG residues initiated by S. cerevisiae AP endonuclease Apn1, which is endowed with a robust progressive 335 exonuclease activity towards duplex DNA. We show that yeast cell extracts, as well as purified Apn1, excise misincorporated 8oxoG, providing a damage-cleansing function to DNA synthesis. Consistent with these results, deletion of both OGG1 encoding 8oxoG-DNA glycosylase and APN1 causes nearly 46-fold synergistic increase in the spontaneous mutation rate, and this enhanced mutagenesis is primarily due to G · C to T · A transversions. Expression of the bacterial 8oxodGTP triphosphotase MutT in the apn1⌬ ogg1⌬ mutant reduces the mutagenesis. Taken together, our results indicate that Apn1 is involved in an S. cerevisiae 8-oxoguanine-DNA glycosylase (Ogg1)-independent repair pathway for 8oxoG residues. Interestingly, the human major AP endonuclease, Ape1, also exhibits similar exonuclease activity towards 8oxoG residues, raising the possibility that this enzyme could participate in the prevention of mutations that would otherwise result from the incorporation of 8oxodGTP.Endogenous oxidative DNA lesions are most abundant and inevitable as cells generate reactive oxygen species through aerobic respiration. The occurrence of oxidized bases in DNA can result from either direct oxidation or misincorporation of oxidized deoxyribonucleoside monophosphate by DNA polymerases from the nucleotide pool (23, 24). These oxidized bases are mutagenic and therefore must be removed by efficient DNA repair processes that include base excision repair (BER) and the nucleotide incision repair (NIR) pathways, as well as by a process involving the sanitization of a pool of oxidized deoxyribonucleotidetriphosphates (dNTPs) (14, 43). In Escherichia coli, three enzymes, formamidopyrimidine-DNA glycosylase (Fpg), adenine-DNA glycosylase (MutY), and a nucleotidase (MutT; 8oxodGTP triphosphatase), play important roles in counteracting the mutagenic effect of the oxidized residue 8-oxo-7,8-dihydrodeoxyguanosine (8oxoG), which forms stable base pairing with adenine (A · 8oxoG) (33). While Fpg excises 8oxoG and MutY excises adenine from the A · 8oxoG mispair in DNA, MutT sanitizes the nucleotide precursor pool of 8oxodGTP by hydrolyzing it to 8oxodGMP and pyrophosphate, thereby preventing the incorporation of the oxidized nucleotide into DNA during replication (12, 32). These three enzymes are also conserved in mammalian cells, thus underscoring the importance of preventing an 8oxoG mutagenic effect (1). However, in the budding yeast Saccharomyces cerevisiae, only a functional homologue of Fpg, Ogg1 (S. cerevisiae 8-oxoguanine-DNA glycosylase), has been is...

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Characterization of Two Independent Amino Acid Substitutions that Disrupt the DNA Repair Functions of the Yeast Apn1

Cited by 24 publications

References 30 publications

Human Glyceraldehyde-3-phosphate Dehydrogenase Plays a Direct Role in Reactivating Oxidized Forms of the DNA Repair Enzyme APE1

Human Glyceraldehyde-3-phosphate Dehydrogenase Plays a Direct Role in Reactivating Oxidized Forms of the DNA Repair Enzyme APE1

A Genome-Wide Screen in Saccharomyces cerevisiae Reveals Altered Transport As a Mechanism of Resistance to the Anticancer Drug Bleomycin

The 3′→5′ Exonuclease of Apn1 Provides an Alternative Pathway To Repair 7,8-Dihydro-8-Oxodeoxyguanosine in Saccharomyces cerevisiae

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