DNA polymerase ζ suppresses the radiosensitivity of glioma cells by regulating the PI3K/AKT/mTOR pathway.

Glioblastoma is the most common glioma with high mortality and poor prognosis. Radiation resistance is one of the large challenges in the treatment of glioma. The study aimed to identify whether DNA polymerase ζ affects glioma cell radiosensitivity. The mRNA and protein levels of REV3L and REV7 were examined using quantitative real-time PCR and western blot. After REV3L and REV7 knockdown in a GBM cell line (A172), we assessed cell viability, colonies, apoptosis, and immune escape. The underlying mechanisms were evaluated using western blot and were confirmed using rescue experiments. The results showed that REV3L and REV7 levels were increased in glioma and related to poor survival. γ-ray treatment inhibited cell viability, survival fraction, and immune escape, and induced apoptosis of glioma cells from a GBM cell line, whereas knockdown of REV3L or REV7 enhanced these effects. Mechanically, silencing of REV3L or REV7 inactivated the PI3K/AKT/mTOR pathway. IGF-1 treatment abrogated the effects on cell viability, colonies, and apoptosis induced by REV3L or REV7 knocking down. Taken together, silencing of REV3L and REV7 inhibited radiation resistance via inactivating the PI3K/AKT/mTOR pathway, suggesting that targeting DNA polymerase ζ may be a new strategy to reduce the radiotherapy resistance of glioma.

Circumventing drug resistance through a CK2-targeted combination via attenuating endogenous AhR-TLS-promoted genomic instability in human colorectal cancer cells.

As anchoring Casein Kinase 2 (CK2) in several human tumors, DN701 as a novel CK2 inhibitor was applied to reverse chemo-resistance via its antitumor effect synergized with oxaliplatin. Recently, translesion DNA synthesis (TLS) has attracted our attention for its association with chemo-resistance, as demonstrated by previous clinical data. The in vitro cell-based properties supported that oxaliplatin combined with DN701 could reverse drug resistance via blockading CK2-mediated aryl hydrocarbon receptor (AhR) and translesion DNA synthesis (TLS)-induced DNA damage repair. Moreover, pharmacologic or genetic inhibition on REV3L (Protein reversion less 3-like) greatly impaired TLS-induced genomic instability. Mechanistically, combination of oxaliplatin with DN701 was found to inhibit CK2 expression and AhR-TLS-REV3L axis signaling, implying the potential decrease of genomic instability. In addition, the combination of oxaliplatin with DN701 could reduce CK2-AhR-TLS-related genomic instability, leading to potent antitumor effects in vivo. Our study presents an underlying mechanism that DN701 could attenuate tumoral chemo-resistance via decaying CK2-mediated AhR and TLS genomic instability, suggesting a potential cancer chemotherapeutic modality to prolong survival in chemo-resistant patients.

Heterogeneity and viral replication fitness of HSV-1 clinical isolates with mutations in the thymidine kinase and DNA polymerase.

BACKGROUND: Prolonged antiviral therapy in immunocompromised individuals can result in the emergence of (multi)drug-resistant herpes simplex virus 1 (HSV-1) infections, forming a therapeutic challenge. OBJECTIVES: To evaluate spatial and temporal differences in drug resistance of HSV-1 samples from a HSCT recipient and to determine the effect of resistance mutations on viral replication fitness. PATIENTS AND METHODS: Five HSV-1 isolates were recovered from a HSCT recipient who suffered from persistent HSV-1 lesions, consecutively treated with aciclovir, foscarnet, cidofovir and a combination of ganciclovir and cidofovir. Spatial and temporal differences in HSV-1 drug resistance were evaluated genotypically [Sanger sequencing and next-generation sequencing (NGS) of the viral thymidine kinase (TK) and DNA polymerase (DP)] and phenotypically (plaque reduction assay). Viral replication fitness was determined by dual infection competition assays. RESULTS: Rapid evolution to aciclovir and foscarnet resistance was observed due to acquisition of TK (A189V and R222H) and DP (L778M and L802F) mutations. Virus isolates showed heterogeneous populations, spatial virus compartmentalization and minor viral variants in three out of five isolates (detectable by NGS but not by Sanger sequencing). Mutations in the TK and DP genes did not alter replication fitness without drug pressure. TK and/or DP mutants influenced replication fitness under antiviral pressure and showed increased fitness under pressure of the drug they showed resistance to. CONCLUSIONS: The use of NGS and dual infection competition assays revealed rapid evolution of HSV-1 drug resistance in a HSCT recipient with spatial and temporal compartmentalization of viral variants that had altered replication fitness under antiviral pressure.

Immediately early 2 (IE-2) and DNA polymerase SiRNA as virus-specific antiviral against novel transplacental cytomegalovirus strain ALL-03 in vitro.

OBJECTIVE: This study investigated the suitability of siRNA targeting specific genes that cause inhibition of virus replication in vitro especially for the virus that capable of crossing placenta and we employed a novel transplacental rat cytomegalovirus that mimics infection in human. METHODS: Six unique siRNAs with three each targeting different regions of IE2 (ie2a, ie2b and ie2c) and DNA polymerase (dpa, dpb and dpc) were prepared and tested for antiviral activities. The efficacy as an antiviral was determined in in-vitro by measuring TCID50 virus titer, severity of virus-induced cytopathic effect (CPE), intracellular viral genome loads by droplet digital PCR, the degree of apoptosis in siRNA-treated cells and relative expression of viral mRNA in infected Rat Embryo Fibroblast (REF) cells. FINDINGS: Remarkably, the siRNAs: dpa, dpb and IE2b, significantly reduced virus yield (approximately >90%) compared to control group at day 18 post infection (p.i). Changes in CPE indicated that DNA polymerase siRNAs were capable of protecting cells against CMV infection at day 14 p.i with higher efficiency than GCV (at the concentration of 300 pmol). Gene expression analysis revealed a marked down regulation of the targeted DNA polymerase gene (73.9%, 96.0% and 90.7% for dpa, dpb and dpc siRNA, respectively) and IE2 gene (50.8%, 49.9% and 15.8% for ie2a, ie2b and ie2c siRNA, respectively) when measured by RT-qPCR. Intracellular viral DNA loads showed a significant reduction for all the DNA polymerase siRNAs (dpa: 96%, dpb: 98% and dpc:92) compared to control group (P < 0.05). CONCLUSION: In conclusion, this study clearly highlighted the feasibility of RNAi as an alternative antiviral therapy that could lead to controlling the CMV infection.

Hepatitis B Virus DNA Polymerase Displays an Anti-Apoptotic Effect by Interacting with Elongation Factor-1 Alpha-2 in Hepatoma Cells.

Hepatitis B virus (HBV) genome P-encoded protein HBV DNA polymerase (Pol) has long been known as a reverse transcriptase during HBV replication. In this study, we investigated the impact of HBV Pol on host cellular processes, mainly apoptosis, and the underlying mechanisms. We showed a marked reduction in apoptotic rates in the HBV Pol-expressed HepG2 cells compared to controls. Moreover, a series of assays, i.e., yeast two-hybrid, GST pull-down, co-immunoprecipitation, and confocal laser scanning microscopy, identified the host factor eEF1A2 to be associated with HBV Pol. Furthermore, knockdown of eEF1A2 gene by siRNA abrogated the HBV Pol-mediated anti-apoptotic effect with apoptosis induced by endoplasmatic reticulum (ER) stress-inducer thapsigargin (TG), thus suggesting that the host factor eEF1A2 is essential for HBV Pol's anti-apoptosis properties. Our findings have revealed a novel role for HBV Pol in its modulation of apoptosis through integrating with eEF1A2.

Switching the activity of Taq polymerase using clamp-like triplex aptamer structure.

In nature, allostery is the principal approach for regulating cellular processes and pathways. Inspired by nature, structure-switching aptamer-based nanodevices are widely used in artificial biotechnologies. However, the canonical aptamer structures in the nanodevices usually adopt a duplex form, which limits the flexibility and controllability. Here, a new regulating strategy based on a clamp-like triplex aptamer structure (CLTAS) was proposed for switching DNA polymerase activity via conformational changes. It was demonstrated that the polymerase activity could be regulated by either adjusting structure parameters or dynamic reactions including strand displacement or enzymatic digestion. Compared with the duplex aptamer structure, the CLTAS possesses programmability, excellent affinity and high discrimination efficiency. The CLTAS was successfully applied to distinguish single-base mismatches. The strategy expands the application scope of triplex structures and shows potential in biosensing and programmable nanomachines.

A Self-Activated Mechanism for Nucleic Acid Polymerization Catalyzed by DNA/RNA Polymerases.

The enzymatic polymerization of DNA and RNA is the basis for genetic inheritance for all living organisms. It is catalyzed by the DNA/RNA polymerase (Pol) superfamily. Here, bioinformatics analysis reveals that the incoming nucleotide substrate always forms an H-bond between its 3'-OH and ß-phosphate moieties upon formation of the Michaelis complex. This previously unrecognized H-bond implies a novel self-activated mechanism (SAM), which synergistically connects the in situ nucleophile formation with subsequent nucleotide addition and, importantly, nucleic acid translocation. Thus, SAM allows an elegant and efficient closed-loop sequence of chemical and physical steps for Pol catalysis. This is markedly different from previous mechanistic hypotheses. Our proposed mechanism is corroborated via ab initio QM/MM simulations on a specific Pol, the human DNA polymerase-η, an enzyme involved in repairing damaged DNA. The structural conservation of DNA and RNA Pols supports the possible extension of SAM to Pol enzymes from the three domains of life.

Thymidylate synthase gene amplification predicts pemetrexed resistance in patients with advanced non-small cell lung cancer

Background. Thymidylate synthase (TYMS) expression in lung cancer tissue affects the therapeutic efficacy of pemetrexed (PMT). TYMS protein expression is primarily assessed using immunohistochemistry (IHC), but this method is not suitable for accurate quantitative analysis. It is not known whether the analysis of TYMS gene copy number using fluorescence in situ hybridization (FISH) is a useful method for assessment of TYMS expression. Patients and methods. The participants were patients with chemo-naïve advanced NSCLC treated with PMT plus carboplatin (CBDCA) in prospective clinical phase II study. TYMS expression was evaluated in 40 patients by gene copy number and protein expression using FISH and IHC. Therapeutic efficacy was evaluated by investigating the response rate (RR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS). Results. TYMS gene amplification was detected in 8 patients (32 %) among 25 patients who could be evaluated for TYMS gene copy number. There were no patients with complete or partial response in the TYMS amplified group. RR and DCR were lower in the TYMS amplified group compared with the TYMS unamplified group (0 versus 35.3 %, p = 0.0539, 62.5 versus 94.1 %, p = 0.0443). PFS and OS were reduced in the TYMS amplified group. The analysis of TYMS gene copy number had higher sensitivity and specificity compared with TYMS protein expression (76.2 versus 50.0 %, 75.0 versus 66.7 %). Conclusion. The analysis of TYMS gene copy number is more suitable than TYMS protein expression for assessment of TYMS expression. TYMS gene amplification predicts outcome of patients receiving PMT with advanced NSCLC (AU)

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Factors affecting the detection and quantification of mitochondrial point heteroplasmy using Sanger sequencing and SNaPshot minisequencing.

Mitochondrial DNA analysis plays an important role in forensic science as well as in the diagnosis of mitochondrial diseases. The occurrence of two different nucleotides at the same sequence position can be caused either by heteroplasmy or by a mix of samples. The detection of superimposed positions in forensic samples and their quantification can provide additional information and might also be useful to identify a mixed sample. Therefore, the detection and visualization of heteroplasmy has to be robust and sensitive at the same time to allow for reliable interpretation of results and to avoid a loss of information. In this study, different factors influencing the analysis of mitochondrial heteroplasmy (DNA polymerases, PCR and sequencing primers, nucleotide incorporation, and sequence context) were examined. BigDye Sanger sequencing and the SNaPshot minisequencing were compared as to the accuracy of detection using artificially created mitochondrial DNA mixtures. Both sequencing strategies showed to be robust, and the parameters tested showed to have a variable impact on the display of nucleotide ratios. However, experiments revealed a high correlation between the expected and the measured nucleotide ratios in cell mixtures. Compared to the SNaPshot minisequencing, Sanger sequencing proved to be the more robust and reliable method for quantification of nucleotide ratios but showed a lower detection sensitivity of minor cytosine components.

Enhanced repair of DNA interstrand crosslinking in ovarian cancer cells from patients following treatment with platinum-based chemotherapy.

Despite high tumour response rates to platinum-based chemotherapy in ovarian cancer survival is poor due to the emergence of drug resistance. Mechanistic studies in clinical material have been hampered by the unavailability of sensitive methods to detect the critical drug-induced effects in individual cells. A modification of the single cell gel electrophoresis (comet) assay allows the sensitive detection of DNA interstrand crosslinking in both tumour and normal cells derived directly from clinical material. Tumour cells isolated from 50 ovarian cancer patients were treated ex vivo with 100 microM cisplatin for 1 h and crosslink formation and repair (unhooking) measured. No significant difference in the peak level of crosslinking in tumour cells was observed between patients who were either newly diagnosed or previously treated with platinum-based therapy, or between tumour and mesothelial cells from an individual patient. This indicates no difference in cellular mechanisms such as drug transport or detoxification. In contrast, the percentage repair (unhooking) of DNA interstrand crosslinks was much greater in the group of treated patients. At 24 h in the 36 newly diagnosed patient tumour samples, only one gave >50% repair and 23 gave <10% repair; however, 19 out of 22 treated patient samples gave >10% repair and 14 showed >50% repair. The estimated median difference (newly diagnosed minus treated) was -52 (95% CI -67 to -28), and the P-value from a Mann-Whitney test was <0.001. In eight patients, it was possible to obtain tumour samples prior to any chemotherapy, and also on relapse or at interval debulking surgery following platinum-based chemotherapy. In these patients, the mean % repair prior to therapy was 2.85 rising to 71.23 following treatment. These data demonstrate increased repair of DNA interstrand crosslinks in ovarian tumour cells following platinum therapy which may contribute to clinical acquired resistance.

Nuevos tratamientos para la infección por virus de la hepatitis C / New treatments for hepatitis C virus infection

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Detection of oxaliplatin-induced DNA crosslinks in vitro and in cancer patients using the alkaline comet assay.

Oxaliplatin is frequently used in the therapy of cancer. In DNA, oxaliplatin induces, like cisplatin, the formation of crosslinks, which are commonly accepted as being responsible for the cytotoxicity of platinum agents. The detection of oxaliplatin-induced DNA crosslink formation and repair could be a good measure of assessing how a patient is responding to the agent. In this study, we used a validated modification of the alkaline comet assay for detecting the presence of these crosslinks in vitro and in cancer patients. The H460 tumour cell line was treated in vitro with a range of oxaliplatin and cisplatin doses, and the subsequent crosslink formation and repair compared between the two agents. In addition, lymphocytes from cancer patients undergoing oxaliplatin-based chemotherapy were assayed for the formation and repair of oxaliplatin-induced crosslinks. A dose-response was observed in the in vitro samples, with cisplatin producing more crosslinks than oxaliplatin at equimolar concentrations and lesions induced by both agents showing different repair efficiencies. Furthermore, evidence of crosslink formation and repair was observed in the peripheral blood lymphocytes of all cancer patients studied, along with the detection of interindividual variability in crosslink formation and repair efficiencies. To the best of our knowledge, this is the first time that oxaliplatin DNA crosslinks have been detected either in vitro or in patient samples using the alkaline comet assay. Due to its sensitivity, rapidity, small cell sample and low cost, the alkaline comet assay is a good method for the detection of oxaliplatin-induced crosslinks and their subsequent repair and, in future clinical studies, could prove to be a valuable tool in assessing/predicting a patient's response to chemotherapy.

Reduced expression of connexin 31.1 in larynx cancer is not caused by GJB5 mutations.

Lack of regular cell-cell interaction is one major cause for neoplastic growth and metastasis. In head and neck squamous cell carcinomas a 10-fold down-regulation of connexin31.1 (GJB5) as well as mutations in the TGF-beta-receptor-II were reported. We performed mutation screenings in GJB5 and the TGF-beta-receptor-II poly(10)adenine hot spot employing larynx cancer samples of 10 patients. Variable length of the TGF-beta-receptor-II adenine homopolymer in controls and tumours indicate a high slippage error rate of the DNA polymerases rendering mutational analyses inconsistent. Lack of GJB5 mutations in the entire tumour collection suggests that this gene is not primarily involved in laryngeal tumorigenesis.

Amplification of plant genomic DNA by Phi29 DNA polymerase for use in physical mapping of the hypermethylated genomic region.

Plant genomes contain a heavily methylated region in which cytosines are methylated in both the symmetrical and asymmetrical sequences. The physical mapping of such a hypermethylated region is difficult because many restriction enzymes are sensitive to methylated cytosine residues in their recognition sites. The Phi29 DNA polymerase provides an efficient and representative amplification of the genomic DNA that is methylation-free. Using this amplified genomic DNA, we were able to show that a heavily methylated genomic DNA region becomes amenable to physical mapping with any restriction enzymes. This protocol will be especially useful for analysis of the heavily methylated region of plant genomes.

Impact of DNA polymerases and their buffer systems on quantitative real-time PCR.

An investigation of the influence of five DNA polymerase-buffer systems on real-time PCR showed that the choice of both DNA polymerase and the buffer system affected the amplification efficiency as well as the detection window. The analytical repeatability of the data for different systems changed clearly, leading us to conclude that basing quantitative measurements on single-data-set standard curves can lead to significant errors.

Bridging PNAs can bind preferentially to a deleted mitochondrial DNA template but replication by mitochondrial DNA polymerase gamma in vitro is not impaired.

Mutations in mitochondrial DNA (mtDNA) are an important cause of neurological and other human pathologies. In the vast majority of cases, supportive care only is available. Mutated and wild-type mtDNAs often coexist in the same cell. A strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Peptide nucleic acid molecules (PNAs) can be designed to selectively target pathogenic mtDNA with single point mutations. Molecules harbouring deletions present a complex problem. Deletions often occur between two short repeat sequences (4-13 residues), one of which is retained in the deleted molecule. With the more common large repeats, it is therefore difficult to design an antigenomic molecule that will bind selectively under physiological conditions. Following limited success with antigenomic oligodeoxynucleotides (ODNs), we have repeated these studies with a series of bridging PNAs. Molecules complementary to the sequence flanking either side of the 13 bp 'common deletion' were synthesised. The PNAs demonstrated markedly greater affinity for the delete than to the wild-type template. In runoff assays using Klenow fragment, these PNAs selectively inhibited replication of the delete template. However, no selective inhibition was observed when a polymerase gamma-containing mitochondrial fraction was used.

Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase iota.

Humans possess four Y-family polymerases: pols eta, iota, kappa and the Rev1 protein. The pivotal role that pol eta plays in protecting us from UV-induced skin cancers is unquestioned given that mutations in the POLH gene (encoding pol eta), lead to the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant phenotype. The roles that pols iota, kappa and Rev1 play in the tolerance of UV-induced DNA damage is, however, much less clear. For example, in vitro studies in which the ability of pol iota to bypass UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone (6-4PP) lesions has been assayed, are somewhat varied with results ranging from limited misinsertion opposite CPDs to complete lesion bypass. We have tested the hypothesis that such discrepancies might have arisen from different assay conditions and local sequence contexts surrounding each UV-photoproduct and find that pol iota can facilitate significant levels of unassisted highly error-prone bypass of a T-T CPD, particularly when the lesion is located in a 3'-A[T-T]A-5' template sequence context and the reaction buffer contains no KCl. When encountering a T-T 6-4PP dimer under the same assay conditions, pol iota efficiently and accurately inserts the correct base, A, opposite the 3'T of the 6-4PP by factors of approximately 10(2) over the incorporation of incorrect nucleotides, while incorporation opposite the 5'T is highly mutagenic. Pol kappa has been proposed to function in the bypass of UV-induced lesions by helping extend primers terminated opposite CPDs. However, we find no evidence that the combined actions of pol iota and pol kappa result in a significant increase in bypass of T-T CPDs when compared to pol iota alone. Our data suggest that under certain conditions and sequence contexts, pol iota can bypass T-T CPDs unassisted and can efficiently incorporate one or more bases opposite a T-T 6-4PP. Such biochemical activities may, therefore, be of biological significance especially in XP-V cells lacking the primary T-T CPD bypassing enzyme, pol eta.

Structure and potential mutagenicity of new hydantoin products from guanosine and 8-oxo-7,8-dihydroguanine oxidation by transition metals.

In vitro work in this laboratory has identified new DNA lesions resulting from further oxidation of a common biomarker of oxidative damage, 8-oxo-7,8-dihydroguanine (OG). The major product of oxidation of OG in a nucleoside, nucleotide, or single-stranded oligodeoxynucleotide using metal ions that act as one-electron oxidants is the new nucleoside derivative spiroiminodihydantoin (Sp). In duplex DNA an equilibrating mixture of two isomeric products, guanidinohydantoin (Gh) and iminoallantoin (Ia), is produced. These products are also formed by the overall four-electron oxidation of guanosine by photochemical processes involving O(2). DNA template strands containing either Sp or Gh/Ia generally acted as a block to DNA synthesis with the Klenow exo(-) fragment of pol I. However, when nucleotide insertion did occur opposite the lesions, only 2'-deoxyadenosine 5-triphosphate and 2'-deoxyguanine 5-triphosphate were used for primer extension. The Escherichia coli DNA repair enzyme Fpg was able to remove the Sp and Gh/Ia lesions from duplex DNA substrates, although the efficiency was depended on the base opposite the lesion.

Guanine and 7,8-dihydro-8-oxo-guanine-specific oxidation in DNA by chromium(V).

The hexavalent oxidation state of chromium [Cr(VI)] is a well-established human carcinogen, although the mechanism of cancer induction is currently unknown. Intracellular reduction of Cr(VI) forms Cr(V), which is thought to play a fundamental role in the mechanism of DNA damage by this carcinogen. Two separate pathways of DNA damage, an oxidative pathway and a metal-binding pathway, have been proposed to account for the lesions observed in cell systems. We have used a model Cr(V) complex, N,N-ethylenebis(salicylidene-animato)oxochromium(V) [Cr(V)-Salen], to investigate the oxidative pathway of DNA damage and to elucidate the lesions generated from this oxidation process. Reaction of Cr(V)-Salen with synthetic oligonucleotides produced guanine-specific lesions that were not 8-oxo-2'-deoxyguanosine, based on the inability of iridium(IV) to further oxidize these sites. Oxidation products were identified using a 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-G) containing oligonucleotide to increase the yields of product for identification by electrospray ionization mass spectrometry. The guanine-based lesions observed by mass spectrometry corresponded to the lesions guanidinohydantoin and spiroiminodihydantoin. The effects of these Cr(V)-Salen-induced lesions on DNA replication fidelity was assayed using a polymerase-based misincorporation assay. These lesions produced G --> T transversion mutations and polymerase stops at levels greater than those observed for 8-oxo-G. These data suggest a model by which chromate can cause DNA damage leading to mutations and cancer.

The S. cerevisiae Mag1 3-methyladenine DNA glycosylase modulates susceptibility to homologous recombination.

DNA glycosylases, such as the Mag1 3-methyladenine (3MeA) DNA glycosylase, initiate the base excision repair (BER) pathway by removing damaged bases to create abasic apurinic/apyrimidinic (AP) sites that are subsequently repaired by downstream BER enzymes. Although unrepaired base damage may be mutagenic or recombinogenic, BER intermediates (e.g. AP sites and strand breaks) may also be problematic. To investigate the molecular basis for methylation-induced homologous recombination events in Saccharomyces cerevisiae, spontaneous and methylation-induced recombination were studied in strains with varied MAG1 expression levels. We show that cells lacking Mag1 have increased susceptibility to methylation-induced recombination, and that disruption of nucleotide excision repair (NER; rad4) in mag1 cells increases cellular susceptibility to these events. Furthermore, expression of Escherichia coli Tag 3MeA DNA glycosylase suppresses recombination events, providing strong evidence that unrepaired 3MeA lesions induce recombination. Disruption of REV3 (required for polymerase zeta (Pol zeta)) in mag1 rad4 cells causes increased susceptibility to methylation-induced toxicity and recombination, suggesting that Pol zeta can replicate past 3MeAs. However, at subtoxic levels of methylation damage, disruption of REV3 suppresses methylation-induced recombination, indicating that the effects of Pol zeta on recombination are highly dose-dependent. We also show that overproduction of Mag1 can increase the levels of spontaneous recombination, presumably due to increased levels of BER intermediates. However, additional APN1 endonuclease expression or disruption of REV3 does not affect MAG1-induced recombination, suggesting that downstream BER intermediates (e.g. single strand breaks) are responsible for MAG1-induced recombination, rather than uncleaved AP sites. Thus, too little Mag1 sensitizes cells to methylation-induced recombination, while too much Mag1 can put cells at risk of recombination induced by single strand breaks formed during BER.