Abstract: 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.

Abstract: We previously isolated the RNC1/TRM2 gene and provided evidence that it encodes a protein with a possible role in DNA double strand break repair. RNC1 was independently re-isolated as the TRM2 gene encoding a methyl transferase involved in tRNA maturation. Here we show that Trm2p purified as a fusion protein displayed 5' --> 3' exonuclease activity on double-strand (ds) DNA, and endonuclease activity on single-strand (ss) DNA, properties characteristic of previously isolated endo-exonucleases. A variant of Trm2p, Trm2p(ctDelta76aa) lacking 76 amino acids at the C-terminus retained nuclease activities but not the methyl transferase activity. Both the native and the variant exhibited sensitivity to the endo-exonuclease inhibitor pentamidine. The Saccharomyces cerevisiae trm2(Delta232-1920nt) mutant (containing only the first 231 nucleotides of the TRM2 gene) displayed low sensitivity to methyl methane sulfonate (MMS) and suppressed the MMS sensitivity of rad52 mutants in trm2(Delta232-1920nt)rad52 double mutants. The deletion of KU80, in trm2(Delta232-1920nt) mutant background displayed higher MMS sensitivity supporting the view of the possible role of Trm2p in a competing repair pathway separate from NHEJ. In addition, trm2 exo1 double mutants were synergistically more sensitive to MMS and ionizing radiation than either of the single mutant suggesting that TRM2 and EXO1 can functionally complement each other. However, the C-terminal portion, required for its methyl transferase activity was found not important for DNA repair. These results propose an important role for TRM2 in DNA repair with a potential involvement of its nuclease function in homologous recombination based repair of DNA DSBs.

Abstract: PTPA, which possesses a peptidyl prolyl isomerase activity, was initially isolated as a protein that stimulates the weak phosphotyrosyl phosphatase activity of the Ser/Thr phosphatase PP2A. Here we show that transient overexpression of PTPA leads to cell death in a time-dependent manner in mammalian cells. PTPA-overproducing cells manifest hallmarks of apoptosis including chromatin condensation, membrane blebbing, positive staining with annexin V, dephosphorylation of Bad, and caspase-3 cleavage. Incubation of cells with the PP2A inhibitor okadaic acid does not prevent either dephosphorylation of Bad or PTPA-induced apoptosis, indicating that PTPA is unlikely to mediate its proapoptotic effect via PP2A. Moreover, we find no evidence for the involvement of either p53 or MAP kinases. Our data reveal a potential novel role for PTPA in the apoptotic process.

Abstract: Reactive oxygen species can attack the mitochondrial genome to produce a vast array of oxidative DNA lesions including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). We assess the role of the Saccharomyces cerevisiae 8-oxo-dGuo DNA glycosylase, Ogg1, in the maintenance of a poly(GT) tract reporter system present in the mitochondrial genome. Deletion in the poly(GT) tract causes the reporter system to produce arginine-independent (Arg+) colonies. We show that the mitochondrial form of Ogg1 is functionally active at processing 8-oxo-dGuo lesions and that Ogg1-deficient cells exhibit nearly six-fold elevated rate of Arg+ mutants under normal growth condition, as compared to the parent. Overexpression of Ogg1 completely suppressed the high rate of Arg+ mutations to levels lower than the parental, suggesting that Ogg1 function could be limited in the mitochondria. Further analysis revealed that the Arg+ mutations can be prevented if the cells are grown under anaerobic conditions. These findings provide in vivo evidence that oxidative stress induces the formation of lesions, most likely 8-oxo-dGuo, which must be repaired by Ogg1, otherwise the lesions can trigger poly(GT) tract instability in the mitochondrial genome. We also demonstrate that overproduction of the major apurinic/apyrimidinic (AP) endonuclease Apn1, a nuclear and mitochondrial enzyme with multiple DNA repair activities, substantially elevated the rate of Arg+ mutants, but which was counteracted by Ogg1 overexpression. We suggest that Ogg1 might bind to AP sites and protect this lesion from the spurious action of Apn1 overproduction. Thus, cleavage of AP site located within or in the vicinity of the poly(GT) tract could destabilize this repeat.

Abstract: We show that mutants lacking either the phosphatase activator Rrd1 or the phosphatase Pph3 are resistant to rapamycin and that double mutants exhibit a synergistic response. This phenotype could be related to an inability of the mutants to degrade RNA polymerase II, leading to transcription of critical genes that sustain growth.

Abstract: The Saccharomyces cerevisiae SPT10 protein possesses a DNA-binding domain that is fused to a putative histone acetyltransferase domain. It binds specifically to upstream-activating sequence elements in the core histone promoters and plays a direct role in histone gene regulation. SPT10 is also required for cell-cycle-specific K56 acetylation at histone genes, allowing the recruitment of the nucleosome remodeling factor Snf5 and subsequent regulation of gene transcription. We reisolated the SPT10 gene in a functional genome-wide screen designed to identify haploid yeast mutants that are hypersensitive to the antitumor drug bleomycin, which acts by damaging DNA. In addition to bleomycin, we show that spt10Delta mutants are also hypersensitive to a limited set of genotoxic agents that create DNA strand breaks, but not to 254-nm ultraviolet light or 4-nitroquinoline-1-oxide, which generate helix distortion. The hypersensitivities of the spt10Delta mutant to the genotoxic agents are rescued by a single copy plasmid carrying the SPT10 gene. We further showed that spt10Delta mutants displayed a modest twofold increase spontaneous mutant frequency, as compared to the parent. Following exposure to bleomycin, these mutants accumulate unrepaired lesions, e.g., DNA strand breaks with blocked 3'-ends in the chromosomal DNA. This defect is not due to the altered expression level or the enzymatic activities of a key DNA repair enzyme, APN1, which is known to repair DNA strand breaks with blocked ends. We propose that SPT10 mediates repair of a subset of DNA lesions by acetylating histones to promote recruitment of DNA repair enzymes.

Abstract: Polyamines play essential functions in many aspects of cell biology. Plasma membrane transport systems for the specific uptake of polyamines exist in most eukaryotic cells but have been very recently identified at the molecular level only in the parasite Leishmania. We now report that the high affinity polyamine permease in Saccharomyces cerevisiae is identical to Agp2p, a member of the yeast amino acid transporter family that was previously identified as a carnitine transporter. Deletion of AGP2 dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines, and transformation with an AGP2 expression vector restored polyamine transport in agp2delta mutants. Yeast mutants deficient in polyamine biosynthesis required >10-fold higher concentrations of exogenous putrescine to restore cell proliferation upon deletion of the AGP2 gene. Disruption of END3, a gene required for an early step of endocytosis, increased the abundance of Agp2p, an effect that was paralleled by a marked up-regulation of spermidine transport velocity. Thus, AGP2 encodes the first eukaryotic permease that preferentially uses spermidine over putrescine as a high affinity substrate and plays a central role in the uptake of polyamines in yeast.

Abstract: The Saccharomyces cerevisiae mutant strain YW778, which lacks apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase DNA repair activities, displays high levels of spontaneous mutations and hypersensitivities to several DNA damaging agents. We searched a cDNA library derived from the nematode Caenorhabditis elegans for gene products that would rescue the DNA repair defects of this yeast mutant. We isolated two genes, apn-1 and exo-3, encoding proteins that have not been previously characterized. Both APN-1 and EXO-3 share significant identity with the functionally established Escherichia coli AP endonucleases, endonuclease IV and exonuclease III, respectively. Strain YW778 expressing either apn-1 or exo-3 shows parental levels of spontaneous mutations, as well as resistance to DNA damaging agents that produce AP sites and DNA single strand breaks with blocked 3'-ends. Using an in vitro assay, we show that the apn-1 and exo-3 genes independently express AP endonuclease activity in the yeast mutant. We further characterize the EXO-3 protein and three of its mutated variants E68A, D190A, and H279A. The E68A variant retains both AP endonuclease and 3'-diesterase repair activities in vitro, yet severely lacks the ability to protect strain YW778 from spontaneous and drug-induced DNA lesions, suggesting that this variant E68A may possess a defect that interferes with the repair process in vivo. In contrast, D190A and H279A are completely devoid of DNA repair activities and fail to rescue the genetic instability of strain YW778. Our data strongly suggest that EXO-3 and APN-1 are enzymes possessing intrinsic AP endonuclease and 3'-diesterase activities.

Abstract: 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 3'-->5' 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 apn1Delta ogg1Delta 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.

Abstract: The Caenorhabditis elegans genes, exo-3 and apn-1, encode the proteins EXO-3 and APN-1, belonging to the exo III and endo IV families of apurinic/apyrimidinic (AP) endonucleases/3'-diesterases, respectively. Homologues of EXO-3 and APN-1 in E. coli and yeast have been clearly documented to repair AP sites and DNA strand breaks with blocked 3' ends to prevent genomic instability. Herein, we purified the C. elegans EXO-3, expressed as a Gst-fusion protein in yeast, and demonstrated that it possesses strong AP endonuclease and 3'-diesterase activities. However, unlike the E. coli counterpart exonuclease III, EXO-3 shows no significant level of 3' --> 5' exonuclease activity following incision at AP sites. In addition, EXO-3 lacks the ability to directly incise DNA at the 5' side of various oxidatively damaged bases, as observed for the human counterpart Ape1, suggesting that C. elegans evolved a member with tailored functions. Importantly, a variant form of EXO-3, E68A, demonstrates altered magnesium-binding properties, and although the in vitro AP endonuclease is nearly fully recovered in the presence of MgCl2, the 3'-diesterase activity is reduced when compared to the native enzyme. We suggest that Glu68 plays a role in coordinating Mg2+ binding for the enzyme catalytic mechanism. Further analysis reveals that neither purified Gst-EXO-3 nor the E68A variant forms a readily detectable DNA-protein complex with an oligonucleotide substrate containing either an AP site or an alpha,beta-unsaturated aldehyde at its 3' end. However, if the reaction is conducted in the presence of crude extracts derived from either yeast or C. elegans embryos, only E68A forms a distinct slow migrating DNA-protein complex with each of the substrates, suggesting that Glu68 may be required to facilitate the release of EXO-3 from the incised DNA to allow entry of the remaining components of the base-excision repair pathway. Thus, the slow migrating DNA-protein complex formed by the E68A variant could be indicative of a stalled repair process with associated factor(s).