Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA.

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.     

Structure/function analysis of the interaction of adenomatous polyposis coli with DNA polymerase beta and its implications for base excision repair

Mutations in the adenomatous polyposis coli (APC) gene are associated with an early onset of colorectal carcinogenesis. Previously, we described a novel role for the APC polypeptide in base excision repair (BER). The single-nucleotide (SN) and long-patch (LP) BER pathways act to repair the abasic sites in DNA that are induced by stressors, such as spontaneous oxidation/reduction, alkylation, and hyperthermia. We have shown that APC interacts with DNA polymerase beta (Pol-beta) and flap endonuclease 1 (Fen-1) and blocks Pol-beta-directed strand-displacement synthesis. In this study, we have mapped the APC interaction site in Pol-beta and have found that Thr79, Lys81, and Arg83 of Pol-beta were critical for its interaction with APC. The Pol-beta protein (T79A/K81A/R83A) blocked strand-displacement DNA synthesis in which tetrahydrofuran was used as DNA substrate. We further showed that the APC-mediated blockage of LP-BER was due to inhibition of Fen-1 activity. Analysis of the APC-mediated blockage of SN-BER indicated that the interaction of APC with Pol-beta blocked SN-BER activity by inhibiting Pol-beta-directed deoxyribose phosphate lyase activity. Collectively, our findings indicate that APC blocked both Pol-beta-directed SN- and LP-BER pathways and increased sensitivity of cells to alkylation induced DNA damage.     

Genetic network interactions among replication, repair and nuclear pore deficiencies in yeast

The yeast RAD27 gene encodes a functional homolog of the mammalian FEN1 protein, a structure-specific endo/exonuclease which plays an important role in DNA replication and repair. Previous genetic interaction studies, including a synthetic genetic array (SGA) analysis, showed that the survival of rad27Delta cells requires several DNA metabolic processes, in particular those mediated by all members of the Rad52-dependent recombinational repair pathway. Here, we report the results of our SGA analysis of the collection of non-essential yeast genes against the rad27Delta mutation, which resulted in the identification of a novel synthetic lethal interaction conferred by mutations affecting the Nup84 nuclear pore subcomplex (nup133Delta, nup120Delta and nup84Delta). Additional screens showed that all Rad52 group genes are required for the survival of the nup133Delta and nup120Delta mutants, which are defective in nuclear pore distribution and mRNA export, but not of the nup133DeltaN mutant, which is solely defective in pore distribution. This requirement for the DNA double-strand break (DSB) repair pathway is consistent with the observation that, like rad27Delta, the nup133Delta, nup120Delta and nup84Delta mutants are sensitive to methyl methanesulfonate (MMS). Furthermore, nup133Delta cells exhibit an increased number of spontaneous DNA repair foci containing Rad52. Altogether, these data suggest that the pathological interactions between the rad27Delta and specific nupDelta mutations result from the accumulation of unrepaired DNA damages.     

Genetic interactions between HNT3/Aprataxin and RAD27/FEN1 suggest parallel pathways for 5′ end processing during base excision repair

Mutations in Aprataxin cause the neurodegenerative syndrome ataxia oculomotor apraxia type 1. Aprataxin catalyzes removal of adenosine monophosphate (AMP) from the 5′ end of a DNA strand, which results from an aborted attempt to ligate a strand break containing a damaged end. To gain insight into which DNA lesions are substrates for Aprataxin action in vivo, we deleted the Saccharomyces cerevisiae HNT3 gene, which encodes the Aprataxin homolog, in combination with known DNA repair genes. While hnt3Δ single mutants were not sensitive to DNA damaging agents, loss of HNT3 caused synergistic sensitivity to H₂O₂ in backgrounds that accumulate strand breaks with blocked termini, including apn1Δ apn2Δ tpp1Δ and ntg1Δ ntg2Δ ogg1Δ. Loss of HNT3 in rad27Δ cells, which are deficient in long-patch base excision repair (LP-BER), resulted in synergistic sensitivity to H₂O₂ and MMS, indicating that Hnt3 and LP-BER provide parallel pathways for processing 5′ AMPs. Loss of HNT3 also increased the sister chromatid exchange frequency. Surprisingly, HNT3 deletion partially rescued H₂O₂ sensitivity in recombination-deficient rad51Δ and rad52Δ cells, suggesting that Hnt3 promotes formation of a repair intermediate that is resolved by recombination.     

Nucleotide excision repair defect influences lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent in the absence of base excision repair

Using a yeast shuttle vector system, we have previously reported on the toxicity and mutagenicity of Me-lex, [1-methyl-4-[1-methyl-4-[3-(methoxysulfonyl)propanamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propane, a compound that selectively generates 3-methyladenine (3-MeA). We observed that a mutant strain defective in Mag1, the glycosylase that excises 3-MeA in the initial step of base excision repair (BER) to generate an abasic site, is significantly more sensitive to the toxicity of Me-lex with respect to wild type but shows only a marginal increase in mutagenicity. A strain defective in AP endonuclease activity (Deltaapn1apn2), also required for functional BER, is equally sensitive to the toxicity as the Deltamag1 mutant but showed a significantly higher mutation frequency. In the present work, we have explored the role of nucleotide excision repair (NER) in Me-lex-induced toxicity and mutagenicity since it is known that NER acts on abasic sites in vivo in yeast and in vitro assays. To accomplish this, we have deleted one of the genes essential for NER in yeast, namely, RAD14, both in the context of an otherwise DNA repair-proficient strain (Deltarad14) and in a BER-defective isogenic derivative lacking the MAG1 gene (Deltamag1rad14). Interestingly, no sensitivity to the treatment with Me-lex was conferred by the simple deletion of RAD14. However, a significant enhancement in toxicity and mutagenicity was observed when cells lacked both Rad14 and Mag1. The mutation spectrum induced by Me-lex in the Deltamag1rad14 strain is indistinguishable from that observed in the Deltaapn1/Deltaapn2 or in the Deltamag1 strains. The results indicate that in yeast NER can play a protective role against 3-MeA-mediated toxicity and mutagenicity; however, the role of NER is appreciable only in a BER-defective background.     

Synergism between base excision repair,mediated by the DNA glycosylases Ntg1 and Ntg2, and nucleotide excision repair in the removal of oxidatively damaged DNA bases in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, inactivation of the two DNA N-glycosylases Ntg1p and Ntg2p does not result in a spontaneous mutator phenotype, whereas simultaneous inactivation of Ntglp, Ntg2p and Radlp or Rad14p, both of which are involved in nucleotide excision repair (NER), does. The triple mutants rad1 ntg1 ntg2 and rad14 ntg1 ntg2 show 15- and 22-fold increases, respectively, in spontaneous forward mutation to canavanine resistance (CanR) relative to the wild-type strain (WT). In contrast, neither of these triple mutants shows an increase in the incidence of Lys+ revertants of the lys1-1 ochre allele. Furthermore, the rad1 ntg1 ntg2 mutant is hypersensitive to the lethal effect of H2O2 relative to WT, rad1 and ntg1 ntg2 mutant strains. Moreover, the rad1 ntg1 ntg2 strain is hypermutable (CanR and Lys+) upon exposure to H2O2, relative to WT, rad1 and ntg1 ntg2 strains. Mutagen sensitivity and enhanced mutagenesis in the rad1 ntg1 ntg2 triple mutant, relative to the other strains tested, were also observed upon exposure to oxidizing agents such as tertbutylhydroperoxide and menadione. In contrast, the sensitivity of the rad1 ntg1 ntg2 triple mutant to gamma-irradiation does not differ from that of the WT. However, the triple mutant shows an increase in the frequency of Lys+ revertants recovered after gamma-irradiation. The results reported in this study demonstrate that base excision repair (BER) mediated by Ntglp and Ntg2p acts synergistically with NER to repair endogenous or induced lethal and mutagenic oxidative DNA damage in yeast. The substrate specificity of Ntg1 p and Ntg2p, and the spectrum of lesions induced by the DNA-damaging agents used, strongly suggest that oxidized DNA bases, presumably oxidized pyrimidines, represent the major targets of this repair pathway.     

MMS1 protects against replication-dependent DNA damage in Saccharomyces cerevisiae

A series of yeast mutants were isolated that are sensitive to killing by the monofunctional DNA-alkylating agent methyl methanesulfonate (MMS) but not by UV or X-radiation. We have cloned and characterized one of the corresponding genes, MMS1, and show that the mms1 Delta mutant is dramatically sensitive to killing by MMS and mildly sensitive to UV radiation. mms1 Delta mutants display an elevated level of spontaneous DNA damage and genomic instability. Furthermore, the mms1 Delta cells are sensitive to killing by conditions that induce replication-dependent double-strand breaks, such as treatment with camptothecin, and incubation of a cdc2-2 strain at the restrictive temperature. rad52 Delta is epistatic to mms1 Delta for MMS and camptothecin sensitivity, indicating that Mms1 acts in concert with Rad52. However, unlike mutants of the RAD52 group, mms1 Delta cells are not sensitive to gamma-rays, which induce double-strand breaks independently of DNA replication. Together these results suggest a role for an Mms1-dependent, Rad52-mediated, pathway in protecting cells against replication-dependent DNA damage.     

Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae

DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to determine whether or not AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We previously reported that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by a model DNA alkylating agent methyl methanesulfonate (MMS) and that this sensitivity can be reduced by deleting the MAG1 3-methyladenine DNA glycosylase gene. Here we report that in the absence of the AP endonucleases, deletion of two Escherichia coli endonuclease III homologs, NTG1 and NTG2, partially suppresses MMS-induced killing, which indicates that the AP lyase products are deleterious unless they are further processed by an AP endonuclease. The severe MMS sensitivity seen in AP endonuclease deficient strains can also be rescued by treatment of cells with the AP lyase inhibitor methoxyamine, which suggests that the product of AP lyase action on an AP site is indeed an extremely toxic lesion. In addition to the AP endonuclease interactions, deletion of NTG1 and NTG2 enhances the mag1 mutant sensitivity to MMS, whereas overexpression of MAG1 in either the ntg1 or ntg2 mutant severely affects cell growth. These results help to delineate alkylation base lesion flow within the BER pathway.     

Complementary functions of the Saccharomyces cerevisiae Rad2 family nucleases in Okazaki fragment maturation,mutation avoidance,and chromosome stability

Rad2 family nucleases, identified by sequence similarity within their catalytic domains, function in multiple pathways of DNA metabolism. Three members of the Saccharomyces cerevisiae Rad2 family, Rad2, Rad27, and exonuclease 1 (Exo1), exhibit both 5' exonuclease and flap endonuclease activities. Deletion of RAD27 results in defective Okazaki fragment maturation, DNA repair, and subsequent defects in mutation avoidance and chromosomal stability. However, strains lacking Rad27 are viable. The expression profile of EXO1 during the cell cycle is similar to that of RAD27 and other genes encoding proteins that function in DNA replication and repair, suggesting Exo1 may function as a back up nuclease for Rad27 in DNA replication. We show that overexpression of EXO1 suppresses multiple rad27 null mutation-associated phenotypes derived from DNA replication defects, including temperature sensitivity, Okazaki fragment accumulation, the rate of minichromosome loss, and an elevated mutation frequency. While generally similar findings were observed with RAD2, overexpression of RAD2, but not EXO1, suppressed the MMS sensitivity of the rad27 null mutant cells. This suggests that Rad2 can uniquely complement Rad27 in base excision repair (BER). Furthermore, Rad2 and Exo1 complemented the mutator phenotypes and cell cycle defects of rad27 mutant strains to differing extents, suggesting distinct in vivo nucleic acid substrates.     

Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Delta and mms2Delta mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase eta (Poleta) but not in the absence of REV3-encoded DNA Polzeta. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.     

A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3′-blocking termini following AP lyase cleavage by Nth1.     

Long patch base excision repair in mammalian mitochondrial genomes

The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5'-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase gamma (pol gamma) and polymerase beta in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5' terminus after strand break would require 5'-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5'-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5'-exo/endonuclease and pol gamma with 3'-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol gamma, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5'-exo/endonuclease in mitochondrial LP-BER.     

The Saccharomyces cerevisiae DNA damage checkpoint is required for efficient repair of double strand breaks by non-homologous end joining

In this work we report that the Saccharomyces cerevisiae RAD9, RAD24, RAD17, MEC1, MEC3 and RAD53 checkpoint genes are required for efficient non-homologous end joining (NHEJ). RAD9 and RAD24 function additionally in this process. Defective NHEJ in rad9Delta-rad24Delta, but not yku80Delta cells, is only partially rescued by imposing G1 or G2/M delays. Thus, checkpoint functions other than transient cell cycle delays may be required for normal levels of NHEJ. Epistasis analysis also indicated that YKU80 and RAD9/RAD24 function in the same pathway for repair of lesions caused by MMS and gamma-irradiation. Unlike NHEJ, the checkpoint pathway is not required for efficient site-specific integration of plasmid DNA into the yeast genome, which is RAD52-dependent, but RAD51-independent.     

Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance

In eukaryotes, the flap endonuclease of Rad27/Fen-1 is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments, and in base excision repair by cleaving a 5' flap structure that may result during base excision repair. Saccharomyces cerevisiae rad27Delta mutants further display a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result from duplications of DNA sequence, indicating a role in mutation avoidance. Two conserved motifs in Rad27/Fen-1 show homology to the 5' --> 3' exonuclease domain of Escherichia coli DNA polymerase I. The strain defective in the 5' --> 3' exonuclease domain in DNA polymerase I shows essentially the same phenotype as the yeast rad27Delta strain. In this study, we expressed the yeast RAD27 gene in an E. coli strain lacking the 5' --> 3' exonuclease domain in DNA polymerase I in order to test whether eukaryotic RAD27/FEN-1 can complement the defect of its bacterial homolog. We found that the yeast Rad27 protein complements sensitivity to methyl methanesulfonate in an E. coli mutant. On the other hand, Rad27 protein did not reduce the high rate of spontaneous mutagenesis in the E. coli tonB gene which results from duplication of DNA. These results indicate that the yeast Rad27 and E. coli 5' --> 3' exonuclease act on the same substrate. We argue that the lack of mutation avoidance of yeast RAD27 in E. coli results from a lack of interaction between the yeast Rad27 protein and the E. coli replication clamp (beta-clamp).     

Interaction between checkpoint genes RAD9, RAD17, RAD24, and RAD53 involved in the determination of yeast Saccharomyces cerevisiae sensitivity to ionizing radiation

Mechanisms for genetic control of cell division cycle (checkpoint control) have been studied in most detail in yeast Saccharomyces cerevisiae. To clarify the role of checkpoint genes RAD9, RAD17, RAD24, and RAD53 in cell radioresistance, double mutants were analyzed for cell sensitivity to ionizing radiation. Double mutants carrying mutations in combination with mutation rad9Delta were shown to manifest the epistatic type of interaction. Our results suggest that checkpoint genes RAD9, RAD17, RAD24, and RAD53 belong to a single epistatic group designated RAD9 and govern the same pathway. Genes RAD9 and RAD53 have a positive effect on sensitivity to gamma-radiation, whereas RAD17 and RAD24 have a negative effect. Interactions between mutations may differ when considering their sensitivity to gamma-radiation and UV light; mutations rad9Delta and rad24Delta were shown to manifest the additive effect in the first case and epistatic effect in the second.     

A new recombinational DNA repair gene from Schizosaccharomyces pombe with homology to Escherichia coli RecA.

A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55(+) (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an M(r) of 38,000. The rhp55Delta mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Delta cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55(+) acts in one DNA repair pathway with rhp51(+) and rhp54(+), homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55(+) is in a different epistasis group for repair of UV-, MMS-, or gamma-ray-induced DNA damage than is rad22(+), a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55(+) is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved.     

Deoxyribophosphate lyase activity of mammalian endonuclease VIII-like proteins

Base excision repair (BER) protects cells from nucleobase DNA damage. In eukaryotic BER, DNA glycosylases generate abasic sites, which are then converted to deoxyribo-5'-phosphate (dRP) and excised by a dRP lyase (dRPase) activity of DNA polymerase beta (Polbeta). Here, we demonstrate that NEIL1 and NEIL2, mammalian homologs of bacterial endonuclease VIII, excise dRP by beta-elimination with the efficiency similar to Polbeta. DNA duplexes imitating BER intermediates after insertion of a single nucleotide were better substrates. NEIL1 and NEIL2 supplied dRPase activity in BER reconstituted with dRPase-null Polbeta. Our results suggest a role for NEILs as backup dRPases in mammalian cells.