Base Excision Repair Intermediates Research Articles

Characterization of an AP endonuclease from sugarcane – ScARP1

Plants are sessile organisms that need to cope with different conditions. The Base Excision Repair (BER) pathway is an important mechanism protecting the genome from DNA lesions. Apurinic/apyrimidinic (AP) endonucleases are key BER enzymes that process AP sites arising either spontaneously or as BER intermediates. In Arabidopsis there are three AP endonucleases: AtARP1, AtAPE1L, and AtAPE2, and in sugarcane two AtARP1 homologues have been identified: ScARP1 and ScARP3. ScARP1 shares 59% sequence identity with Arabidopsis AtARP. Protein modeling of ScARP1 and AtARP1 revealed conserved active sites and metal binding sites. For biochemical characterisation, recombinant ScARP1 protein displayed AP endonuclease activity both in the presence of MnCl2 or MgCl2 and the optimal temperature for its activity was 37 °C. Under these conditions, 3′-exonuclease, 3′-phosphatase, and 3′-phosphodiesteterase activities were not detectable. We also show that ScARP1 protein is able to complement mutant atarp−/− cell extracts deficient in AP endonuclease activity. These results suggest that AP endonucleases from different plant species preserve AP endonuclease activity. The biochemical characterisation of ScARP1 extends our knowledge of the BER pathway to a monocot crop plant group.

The lyase activity of bifunctional DNA glycosylases and the 3'-diesterase activity of APE1 contribute to the repair of oxidized bases in nucleosomes.

The vast majority of oxidized bases that form in DNA are subject to base excision repair (BER). The DNA intermediates generated during successive steps in BER may prove mutagenic or lethal, making it critical that they be ‘handed’ from one BER enzyme to the next in a coordinated fashion. Here, we report that the handoff of BER intermediates that occurs during the repair of naked DNA substrates differs significantly from that in nucleosomes. During BER of oxidized bases in naked DNA, products generated by the DNA glycosylase NTHL1 were efficiently processed by the downstream enzyme, AP-endonuclease (APE1). In nucleosomes, however, NTHL1-generated products accumulated to significant levels and persisted for some time. During BER of naked DNA substrates, APE1 completely bypasses the inefficient lyase activity of NTHL1. In nucleosomes, the NTHL1-associated lyase contributes to BER, even in the presence of APE1. Moreover, in nucleosomes but not in naked DNA, APE1 was able to process NTHL1 lyase-generated substrates just as efficiently as it processed abasic sites. Thus, the lyase activity of hNTHL1, and the 3′ diesterase activity of APE1, which had been seen as relatively dispensable, may have been preserved during evolution to enhance BER in chromatin.

Abstract 2819: Targeting histone chaperone FACT complex sensitizes medulloblastoma cells to chemo and radiation therapy

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children. Post-surgical radiotherapy in combination with adjuvant chemotherapy are considered as standard treatment of care for MB. However, this long-term treatment often leads to harmful neurologic effects to the developing brain in children. Therefore, identification of novel therapeutic target to increase efficacy and reduce toxicity in MB represent an unmet clinical need. Apurinic/apyrimidinic (AP)-endonuclease (APE1) plays a central role in the base excision repair (BER) pathway by repairing AP sites and single-strand breaks, the downstream cytotoxic BER intermediate that occurs following radiation and chemotherapy. Elevated levels of APE1 or its activity has been shown to be associated with response to radiation and chemotherapy in MB. However, how APE1 repairs AP sites in the context of nucleosome in cells and how APE1-dependnt BER pathway can be manipulated to increase the therapeutic efficacy of chemo and radiation therapy remain an open question. We have recently shown that APE1 is acetylated (AcAPE1) at AP site damage in chromatin by p300 and that acetylation enhances AP-endonuclease activity of APE1. Here, we show that APE1 interacts with the nucleosome remodeling histone chaperone Facilitates of Chromatin Transcription (FACT) complex (a heterodimer of SSRP1 and SPT16 proteins) and that FACT complex is required for binding and acetylation of APE1 at AP site damages in highly aggressive MB cell lines HD-MB03 and ONS-76. Furthermore, we have mapped genome-wide binding of AcAPE1 in HDMB cells and our ChIP-seq data analysis revealed ~68154 (p<.001) AcAPE1 binding predominantly located in the transcribed gene regions corresponding to 16370 genes. We aligned ChIP-Seq with RNA-Seq of HD-MB03 cells and found a significant (p<0.0001) correlation of AcAPE1 binding with gene expression. Furthermore, our data show that inhibition of FACT with small molecule inhibitor CBL0137/curaxin, reduces SSRP1 and SPT16 levels from soluble fraction and trap them in chromatin. We found that CBL0137/curaxin treatment results in inhibition of APE1 acetylation, and that MB cells cannot repair DNA damages in the presence of FACT inhibitor. FACT has been shown to play an important role in removal of cisplatin-induced DNA adducts via nucleotide excision repair pathway (NER). Of importance, downregulation of APE1 levels or inhibition of FACT with CBL0137/curaxin sensitizes a panel of highly aggressive MB cell lines to cisplatin and alkylating drug temozolomide. Together, our study suggest that co-suppression of both BER and NER pathways via targeting FACT complex can greatly sensitize MB tumors to chemo and radiation therapy. A major implication of our studies is that combination of curaxin with low dose of cisplatin may have considerable therapeutic potential to improve efficacy and reduce cytotoxic side effects of cisplatin to MB patients. Citation Format: Sutapa Ray, Heyu Song, Shrabasti Roychoudhury, Pranjal Biswas, Sutapa Ray, Kishor Bhakat. Targeting histone chaperone FACT complex sensitizes medulloblastoma cells to chemo and radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2819.

Nitric oxide induced S-nitrosation causes base excision repair imbalance

It is well established that inflammation leads to the creation of potent DNA damaging chemicals, including reactive oxygen and nitrogen species. Nitric oxide can react with glutathione to create S-nitrosoglutathione (GSNO), which can in turn lead to S-nitrosated proteins. Of particular interest is the impact of GSNO on the function of DNA repair enzymes. The base excision repair (BER) pathway can be initiated by the alkyl-adenine DNA glycosylase (AAG), a monofunctional glycosylase that removes methylated bases. After base removal, an abasic site is formed, which then gets cleaved by AP endonuclease and processed by downstream BER enzymes. Interestingly, using the Fluorescence-based Multiplexed Host Cell Reactivation Assay (FM-HCR), we show that GSNO actually enhances AAG activity, which is consistent with the literature. This raised the possibility that there might be imbalanced BER when cells are challenged with a methylating agent. To further explore this possibility, we confirmed that GSNO can cause AP endonuclease to translocate from the nucleus to the cytoplasm, which might further exacerbate imbalanced BER by increasing the levels of AP sites. Analysis of abasic sites indeed shows GSNO induces an increase in the level of AP sites. Furthermore, analysis of DNA damage using the CometChip (a higher throughput version of the comet assay) shows an increase in the levels of BER intermediates. Finally, we found that GSNO exposure is associated with an increase in methylation-induced cytotoxicity. Taken together, these studies support a model wherein GSNO increases BER initiation while processing of AP sites is decreased, leading to a toxic increase in BER intermediates. This model is also supported by additional studies performed in our laboratory showing that inflammation in vivo leads to increased large-scale sequence rearrangements. Taken together, this work provides new evidence that inflammatory chemicals can drive cytotoxicity and mutagenesis via BER imbalance.

PARP1 and PARP2 stabilise replication forks at base excision repair intermediates through Fbh1-dependent Rad51 regulation

PARP1 regulates the repair of DNA single-strand breaks generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whereas PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication-associated DNA damage owing to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection. Together, our results demonstrate how PARP1 and PARP2 regulate two independent, but intrinsically linked aspects of DNA base damage tolerance by promoting BER directly, and by stabilising replication forks that encounter BER intermediates.

ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair.

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1’s ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1’s role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.

Phaeocystis globosa Virus DNA Polymerase X: a \u201cSwiss Army knife\u201d, Multifunctional DNA polymerase-lyase-ligase for Base Excision Repair

Phaeocystis globosa virus 16T is a giant virus that belongs to the so-called nucleo-cytoplasmic large DNA virus (NCLDV) group. Its linear dsDNA genome contains an almost full complement of genes required to participate in viral base excision repair (BER). Among them is a gene coding for a bimodular protein consisting of an N-terminal Polβ-like core fused to a C-terminal domain (PgVPolX), which shows homology with NAD+-dependent DNA ligases. Analysis of the biochemical features of the purified enzyme revealed that PgVPolX is a multifunctional protein that could act as a “Swiss army knife” enzyme during BER since it is endowed with: 1) a template-directed DNA polymerization activity, preferentially acting on DNA structures containing gaps; 2) 5′-deoxyribose-5-phosphate (dRP) and abasic (AP) site lyase activities; and 3) an NAD+-dependent DNA ligase activity. We show how the three activities act in concert to efficiently repair BER intermediates, leading us to suggest that PgVPolX may constitute, together with the viral AP-endonuclease, a BER pathway. This is the first time that this type of protein fusion has been demonstrated to be functional.

Complementation of aprataxin deficiency by base excision repair enzymes in mitochondrial extracts.

Mitochondrial aprataxin (APTX) protects the mitochondrial genome from the consequence of ligase failure by removing the abortive ligation product, i.e. the 5′-adenylate (5′-AMP) group, during DNA replication and repair. In the absence of APTX activity, blocked base excision repair (BER) intermediates containing the 5′-AMP or 5′-adenylated-deoxyribose phosphate (5′-AMP-dRP) lesions may accumulate. In the current study, we examined DNA polymerase (pol) γ and pol β as possible complementing enzymes in the case of APTX deficiency. The activities of pol β lyase and FEN1 nucleotide excision were able to remove the 5′-AMP-dRP group in mitochondrial extracts from APTX−/− cells. However, the lyase activity of purified pol γ was weak against the 5′-AMP-dRP block in a model BER substrate, and this activity was not able to complement APTX deficiency in mitochondrial extracts from APTX−/−Pol β−/− cells. FEN1 also failed to provide excision of the 5′-adenylated BER intermediate in mitochondrial extracts. These results illustrate the potential role of pol β in complementing APTX deficiency in mitochondria.

5-Formyl- and 5-Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells.

5-Formyl-dC (fdC) and 5-carboxy-dC (cadC) are newly discovered bases in the mammalian genome that are supposed to be substrates for base excision repair (BER) in the framework of active demethylation. The bases are recognized by the monofunctional thymine DNA glycosylase (Tdg), which cleaves the glycosidic bond of the bases to give potentially harmful abasic sites (AP-sites). Because of the turnover of fdC and cadC during cell state transitions, it is an open question to what extent such harmful AP-sites may accumulate during these processes. Here, we report the development of a new reagent that in combination with mass spectrometry (MS) allows us to quantify the levels of AP-sites. This combination also allowed the quantification of β-elimination (βE) products, which are repair intermediates of bifunctional DNA glycosylases. In combination with feeding of isotopically labeled nucleosides, we were able to trace the intermediates back to their original nucleobases. We show that, while the steady-state levels of fdC and cadC are substantially increased in Tdg-deficient cells, those of both AP- and βE-sites are unaltered. The levels of the detected BER intermediates are 1 and 2 orders of magnitude lower than those of cadC and fdC, respectively. Thus, neither the presence of fdC nor that of cadC in stem cells leads to the accumulation of harmful AP- and βE-site intermediates.

Abstract B26: Development of a proximity ligation assay for the detection of PARP-1 trapped to chromatin

Abstract Background: PARP inhibitors (PARPi) have been under clinical investigation as anti-cancer therapies both as monotherapy and in combination. The two basic mechanisms underlying PARP inhibitor activity are catalytic inhibition and PARP-1 trapping. PARPi trap PARP-1 onto base excision repair intermediates, contributing to potentiation of DNA damage by alkylating agents. The role of PARP-1 trapping with regard to efficacy and tolerability in the clinic is still under investigation. All PARPis are capable of PARP-1 trapping; however, different clinical-stage PARPi trap with dramatically different efficiencies. High efficiency trapping agents trap at doses similar to those required for catalytic inhibition while low efficiency trapping agents trap only at doses much higher than those required for inhibition. Being able to image trapped PARP-1 complexes in the cellular context would be beneficial for identifying other proteins associated with these complexes and may have clinical application in the detection of trapped PARP in tumor biopsies. To date, detection of PARP-1 complexes has been limited to low through-put, techniques that disrupt cellular architecture and lack the sensitivity to detect PARP-1 trapping in the absence of supraphysiologic doses of alkylating agents. More sensitive methods for quantifying trapped PARP1 complexes with spatial resolution are needed to enable further understanding of PARP-1 trapping in cancer and normal cells. The objective of this study was to develop an assay capable of identifying cellular PARP-1 trapped to chromatin with enhanced sensitivity and higher assay throughput. Methods: Duolink is a 96-well plate adaptable system that uses a proprietary antibody/oligonucleotide conjugate that, when localized within 40 angstroms, will anneal, amplify and hybridize to generate a quantitative proximity ligation assay (PLA) signal. To develop a Duolink trapping assay, antibodies to PARP-1, histone H2A.X and phosphorylated (Ser139) H2A.X were identified and optimized for labeling concentrations, endogenous target protein recognition and suitability for high content imaging. Duolink PLA was performed per the manufacturer's protocol. Automated image acquisition by Cell Insight and data analysis were performed using Cellomics View Software. PARP1 trapping was also evaluated by cellular fractionation and Western blot. Results: Binding to chromatin places PARP-1 in close proximity to histone H2A.X, presumably within the distance required for successful Duolink oligonucleotide annealing and amplification reactions. Co-treating cells with alkylating agents and PARPi induces a significant increase in phosphorylation of H2A.X (Ser139) as well as trapped PARP1 complexes. Comparative analysis of Western blot and PLA signals in HeyA8 cells treated with different PARPi/alkylating agent combinations revealed positive rank-order correlation in trapping activity. Resolution of in vitro single agent PARP-1 trapping by Western blot has been elusive but PLA detection of single agent PARP1 trapping was observed. Conclusion: The PLA trapping assay provides an improved methodology for evaluation of endogenous PARP-1 complexes bound to chromatin and may have utility for clinical tissue specimens where detection of PARP-1 trapping may lead to insights regarding efficacy and tolerability of PARP inhibitors. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Todd Hopkins, Barrett Ainsworth, Vivek Abraham, David Maag, Eric Johnson, Julie Wilsbacher. Development of a proximity ligation assay for the detection of PARP-1 trapped to chromatin [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B26.

Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair.

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined.

An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation.

Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM.

The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability, but Not for DNA Methylation in Neurospora crassa.

LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to DNA damaging agents. MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued DNA damage-hypersensitivity of Δmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Δmus-30 is partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Δmus-30 strains. We found that MUS-30-deficient cells are not defective for DSB repair, and we observed a negative genetic interaction between Δmus-30 and Δmei-3, the Neurospora RAD51 homolog required for homologous recombination. Together, our findings suggest that MUS-30, an LSH/DDM1 homolog, is required to prevent DNA damage arising from toxic base excision repair intermediates. Overall, our study provides important new information about the functions of the LSH/DDM1 family of enzymes.

P21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates.

The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.

3CAPS - a structural AP-site analogue as a tool to investigate DNA base excision repair.

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP–sites. With its 3′–phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′–deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

Abstract C52: PARP1 trapping activity of PARP inhibitors is associated with cytotoxicity in both cancer cells and healthy bone marrow

Abstract Background: PARP inhibitors (PARPi) are under clinical investigation as anticancer therapies in several indications with a focus on monotherapy in tumors with homologous recombination deficits and on combination regimens with DNA damaging chemotherapy. The combination activity of PARPi with DNA alkylating agents is due in part to trapping of PARP1 onto base excision repair intermediates. Recent studies in mice indicate that potent trapping activity limits the tolerability of PARPi in this setting. While trapping has not been directly demonstrated with PARP inhibitors alone, genetic and correlative data suggest that it may be important in the monotherapy setting as well. The objective of this study was to further delineate the role of trapping in single-agent cytotoxicity of PARPi in cancer cells and healthy bone marrow. Methods: Cytotoxic potency was determined for cancer cells using CellTiter-Glo and for erythroid and myeloid progenitors using colony forming unit (CFU) assays. PARP1 trapping was evaluated by cellular fractionation, TR-FRET and BLI. PARPi binding affinities and off-rates were determined via SPR. Cellular PAR levels were determined by ELISA. Results: Talazoparib, rucaparib and the novel PARPi A-934935 bound to PARP1 with similar affinities and off-rates and inhibited cellular PAR synthesis with identical potencies. Despite these similarities, talazoparib was a more potent trapping agent. Consistent with the increased trapping activity, talazoparib was >10-fold more potent than rucaparib and A-934935 at inhibiting proliferation of erythroid progenitors and >50-fold more potent in myeloid progenitors. Similar differences were observed in a panel of human cancer cell lines including BRCA−/− breast cancer and small cell lung cancer. Conclusions: All NAD+-competitive PARPi elicit PARP1 trapping, but different inhibitors display varying degrees of resolution between trapping and cellular PAR depletion. Understanding the relative importance of these mechanisms to the tolerability and efficacy of PARP inhibitors is of critical importance. Myelosuppression is a commonly observed toxicity in PARPi clinical trials. In the present study we used ex vivo bone marrow CFU assays to model myelosuppression. The IC50s observed were within the range of exposures observed at recommended phase II doses in monotherapy clinical trials, highlighting the clinical relevance of this experimental approach. Our data reveal that compounds inhibiting cellular PAR synthesis with equal potency span a broad range of potency in BM-CFU assays in a manner predicted by PARP1 trapping activity. These results suggest that PARP1 trapping is a primary driver of myelosuppression by PARP inhibitors. Our observation that trapping drives cytotoxicity to a similar extent in bone marrow and cancer cells sensitive to single-agent PARP inhibition suggests that PARP1 trapping activity is not advantageous with respect to the therapeutic index of PARPi in a monotherapy setting. This is consistent with observations related to the therapeutic index of PARP inhibitors in the clinic. These results also have important implications for selection of a PARPi for combination regimens where trapping is not required for efficacy and in which myelosuppression may be a concern, such as those containing platinum drugs or topoisomerase I inhibitors. Citation Format: Todd A. Hopkins, Julie L. Wilsbacher, Enrico L. DiGiammarino, Sanjay C. Panchal, Gui-Dong Zhu, Thomas D. Penning, Eric F. Johnson, David Maag. PARP1 trapping activity of PARP inhibitors is associated with cytotoxicity in both cancer cells and healthy bone marrow. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C52.

Reprint of "Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair".

DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER.

Impact of topical application of sulfur mustard on mice skin and distant organs DNA repair enzyme signature.

Sulfur mustard (SM) is a chemical warfare agent that, upon topical application, damages skin and reaches internal organs through diffusion in blood. Two major toxic consequences of SM exposure are inflammation, associated with oxidative stress, and the formation of alkylated DNA bases. In the present study, we investigated the impact of exposure to SM on DNA repair, using two different functional DNA repair assays which provide information on several Base Excision Repair (BER) and Excision/Synthesis Repair (ESR) activities. BER activities were reduced in all organs as early as 4h after exposure, with the exception of the defense systems against 8-oxo-guanine and hypoxanthine which were stimulated. Interestingly, the resulting BER intermediates could activate inflammation signals, aggravating the inflammation triggered by SM exposure and leading to increased oxidative stress. ESR activities were found to be mostly inhibited in skin, brain and kidneys. In contrast, in the lung there was a general increase in ESR activities. In summary, exposure to SM leads to a significant decrease in DNA repair in most organs, concomitant with the formation of DNA damage. These synergistic genotoxic effects are likely to participate in the high toxicity of this alkylating agent. Lungs, possibly better equipped with repair enzymes to handle exogenous exposure, are the exception.

Abstract 3028: Tolerance and intolerance of proximal thymine glycol in DNA double-strand break repair by nonhomologous end joining

Abstract Double-strand breaks (DSBs) induced by ionizing radiation are often accompanied by ancillary oxidative base damage that could prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, plasmid DSB substrates were constructed with two blunt ends, one having the oxidatively modified nonplanar base thymine glycol at the first, second or third positions from the 3′ terminus (Tg1, Tg2 and Tg3, respectively). End joining was examined in extracts of Bustel fibroblasts, wherein end joining is completely dependent on addition of exogenous recombinant XLF, and therefore reflects classical nonhomologous end joining. Tg at the third position had almost no effect on end-joining even when present on both ends of the break. However, the Tg1 and Tg2 substrates yielded tenfold to twentyfold less head-to-tail joins and no detectable head-to-head joins, suggesting that Tg as the terminal or penultimate base is a serious barrier to NHEJ and an absolute barrier when present at both ends. For all substrates, Tg-containing ligation products predominated at early times, and remarkably, a 3′ terminal Tg was in some cases directly ligated to an undamaged blunt end. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised in the extracts, but largely if not exclusively after DSB ligation. For all substrates, accurate blunt end ligation without end trimming predominated. However addition of excess Artemis nuclease to the extracts resulted in a slightly shorter ligation product, and a slight increase in yield, for the Tg2 substrate only. For the Tg1 substrate, there was a distinct 2-hr delay between addition of substrate and the detection of ligated products, while the unmodified and Tg3 products showed a much shorter delay of ∼30 min. Overall, the results suggest that Tg can be a major impediment to DSB repair when present as the terminal or penultimate base, being relatively resistant to both trimming and ligation. Citation Format: Duaa Bafail, Sri Lakshmi Chalasani, Mohammed Al mohaini, Konstantin Akopiants, Lawrence F. Povirk. Tolerance and intolerance of proximal thymine glycol in DNA double-strand break repair by nonhomologous end joining. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3028. doi:10.1158/1538-7445.AM2015-3028

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with apurinic/apyrimidinic sites in DNA

Apurinic/apyrimidinic (AP) sites are some of the most frequent DNA damages and the key intermediates of base excision repair. Certain proteins can interact with the deoxyribose of the AP site to form a Schiff base, which can be stabilized by NaBH4 treatment. Several types of DNA containing the AP site were used to trap proteins in human cell extracts by this method. In the case of single-stranded AP DNA and AP DNA duplex with both 5′ and 3′ dangling ends, the major crosslinking product had an apparent molecular mass of 45kDa. Using peptide mass mapping based on mass spectrometry data, we identified the protein forming this adduct as an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) called “uracil-DNA glycosylase”. GAPDH is a glycolytic enzyme with many additional putative functions, which include interaction with nucleic acids, different DNA damages and DNA repair enzymes. We investigated interaction of GAPDH purified from HeLa cells and rabbit muscles with different AP DNAs. In spite of the ability to form a Schiff-base intermediate with the deoxyribose of the AP site, GAPDH does not display the AP lyase activity. In addition, along with the borohydride-dependent adducts with AP DNAs containing single-stranded regions, GAPDH was also shown to form the stable borohydride-independent crosslinks with these AP DNAs. GAPDH was proven to crosslink preferentially to AP DNAs cleaved via the β-elimination mechanism (spontaneously or by AP lyases) as compared to DNAs containing the intact AP site. The level of GAPDH–AP DNA adduct formation depends on oxidation of the protein SH-groups; disulfide bond reduction in GAPDH leads to the loss of its ability to form the adducts with AP DNA. A possible role of formation of the stable adducts with AP sites by GAPDH is discussed.