Direct Damage Reversal Research Articles

Base flipping mechanism and binding strength of methyl-damaged DNA during the interaction with AGT.

Methyl damage to DNA bases is common in the cell nucleus. O6-alkylguanine-DNA alkyl transferase (AGT) may be a promising candidate for direct damage reversal in methylated DNA (mDNA) at the O6 point of the guanine. Indeed, atomic-level investigations in the contact region of AGT-DNA complex can provide an in-depth understanding of their binding mechanism, allowing to evaluate the silico-drug nature of AGT and its utility in removing methyl damage in DNA. In this study, molecular dynamics (MD) simulation was utilized to examine the flipping of methylated nucleotide, the binding mechanism between mDNA and AGT, and the comparison of binding strength prior and post methyl transfer to AGT. The study reveals that methylation at the O6 atom of guanine weakens the hydrogen bond (H-bond) between guanine and cytosine, permitting for the flipping of such nucleotide. The formation of a H-bond between the base pair of methylated nucleotide (i.e., cytosine) and the intercalated arginine of AGT also forces the nucleotide to rotate. Following that, electrostatics and van der Waals contacts as well as hydrogen bonding contribute to form the complex of DNA and protein. The stronger binding of AGT with DNA before methyl transfer creates the suitable condition to transfer methyl adduct from DNA to AGT.

DNA Alkylation Damage by Nitrosamines and Relevant DNA Repair Pathways

Nitrosamines occur widespread in food, drinking water, cosmetics, as well as tobacco smoke and can arise endogenously. More recently, nitrosamines have been detected as impurities in various drugs. This is of particular concern as nitrosamines are alkylating agents that are genotoxic and carcinogenic. We first summarize the current knowledge on the different sources and chemical nature of alkylating agents with a focus on relevant nitrosamines. Subsequently, we present the major DNA alkylation adducts induced by nitrosamines upon their metabolic activation by CYP450 monooxygenases. We then describe the DNA repair pathways engaged by the various DNA alkylation adducts, which include base excision repair, direct damage reversal by MGMT and ALKBH, as well as nucleotide excision repair. Their roles in the protection against the genotoxic and carcinogenic effects of nitrosamines are highlighted. Finally, we address DNA translesion synthesis as a DNA damage tolerance mechanism relevant to DNA alkylation adducts.

Abstract 1741: Identification of microsatellite instability in hematologic malignant cell lines

Abstract Defective DNA repair machinery which is a consequence of chromosomal or molecular alterations is associated with variety of diseases including solid tumors. Cytogenetic analysis is an important tool in diagnosis and treatment of hematologic malignancies. Our aim is to understand the role of microsatellite instability in initiation and progression of blood cancers. Here we hypothesize that leukemogenic cells may carry defective repair mechanisms that contribute to other genomic instabilities. In order to explore the possible role of microsatellite instability (MSI) in leukemia and lymphoma, eight different DNA repair genes involved in mismatch repair (MMR) and direct reversal of damage (DRD) were analyzed in K562 (CML), Molt-4 (T-ALL) and Daudi (Burkitt’s lymphoma) cell lines. Fragment analysis was carried out using novel primers considering repeat motifs enriched with (A)n, (CA)n and (AT)n for instability. Almost all the cell lines showed moderate to high level of microsatellite instability and exhibited a shift in selected repeat regions indicating possible loss (66.7%) (MSH2, MSH4, MSH6, MLH1, MLH3, PMS1) or gain (25%) (MSH3, MGMT) of nucleotide repeats as compared to normal DNA. The K562, Philadelphia negative CML cell line appeared to be microsatellite stable (MSS) for genes MSH2 and MLH1. Treatment induced microsatellite instability is known in leukemic patient cells as also seen in Molt-4 cells. Our present and previous observations suggest that MSI is a non-random event in leukemia/lymphoma which needs confirmatory future studies in newly diagnosed untreated patient samples. Considering the fact that both MSI and MSS represents two distinct molecular pathways of carcinogenesis, assessment of MSI, if added as distinct diagnostic criteria, can possibly refine early detection and help in identifying novel targets in management of hematologic malignancies. Citation Format: Priyanjali Bhattacharya, Naveed Anjum Chikan, Arati Sharma, Dhimant Desai, Charyguly Annageldiyev, Pinaki Patel, Trupti N. Patel. Identification of microsatellite instability in hematologic malignant cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1741.

Microsatellite Instability and Promoter Hypermethylation of DNA repair genes in Hematologic Malignancies: a forthcoming direction toward diagnostics

ABSTRACTObjective: The objective of our review is to highlight the significance of microsatellite hypervariation in diagnostics of hematologic malignancies.Methods: For the past few decades, extensive experiments in cancer research have explored all the possible pathways and a number of deleterious mutations that either make the tumor suppressor genes (TSGs) dysfunctional or cause the proto-oncogenes to behave abnormally by changing the cellular phenotype hence rendering disease. To prevent the deleterious effects of mutations and to protect the genomic integrity, our system possesses multiple repair mechanisms. DNA Mismatch Repair (MMR) and Direct Reversal of Damage (DRD) are two repair mechanisms which help in removal of faulty base pairs and alkyl adduct formation respectively to avoid long term effects of toxicity, tumorigenesis and mutagenesis. There are nine major MMR genes – MutS homolog (MSH2, MSH3, MSH4, MSH5, MSH6), MutL homolog (MLH1, MLH3), human post-meiotic segregation genes (PMS1, PMS2), and three major damage reversal genes – O6-methylguanine-DNA-methyltransferase (MGMT), ABH2 and DEPC1.Results: Any malfunction in DNA repair machinery can cause microsatellite instability (MSI), a form of genomic abnormality with hyper mutable repeats that is directly associated with cancer. Microsatellites are short, repetitive sequences, non-randomly distributed and localized in 3'-UTR (Untranslated Region), introns, coding regions and promoters. Besides MSI, evidence on promoter hypermethylation of selected repair genes also points toward a prominent reason for cancer initiation and progression.Conclusion: The presence of specific microsatellite marker hyper-mutability and consistent promoter hypermethylation in leukemia or lymphoma can be considered as a part of routine diagnostic test in clinical laboratories.

Recognition and repair of chemically heterogeneous structures at DNA ends.

Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not "clean." Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini.

Highly Mutagenic Exocyclic DNA Adducts Are Substrates for the Human Nucleotide Incision Repair Pathway

BackgroundOxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to the formation of etheno(ε)-bases including 1,N 6-ethenoadenine (εA) and 3,N 4-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER) pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR) pathway, the apurinic/apyrimidinic (AP) endonucleases can directly incise DNA duplex 5′ to a damaged base in a DNA glycosylase-independent manner.Methodology/Principal FindingsHere we have characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg) and 7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. Activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has a high affinity for DNA containing ε-bases but cleaves DNA duplexes at an extremely slow rate. Consistent with this observation, oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC50 values in the range of 5–10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εA•T and εC•G duplexes generates, as expected, DNA fragments containing 5′-terminal ε-base residue.Conclusions/SignificanceThe fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggests that NIR may act as a backup pathway to BER to remove a large variety of genotoxic base lesions in human cells.

Effects of 5′,8-Cyclodeoxyadenosine Triphosphates on DNA Synthesis

Hydroxyl radicals generate a broad range of DNA lesions in living cells. Cyclopurine deoxynucleosides (CPUs) are a biologically significant class of oxidative DNA lesions due to their helical distortion and chemically stability. The CPUs on DNA are substrates for the nucleotide excision repair (NER) but not for base excision repair or direct damage reversal. Moreover, these lesions block DNA and RNA polymerases, resulting in cell death. Here, we describe the chemical synthesis of 5'S and 5'R isomers of 5',8-cyclodeoxyadenosine triphosphate (cdATP) and demonstrate their ability to be incorporated into DNA by replicative DNA polymerases. DNA synthesis assays revealed that the incorporation of the stereoisomeric cdATPs strongly inhibits DNA polymerase reactions. Surprisingly, the two stereoisomers had different mutagenic profiles, since the S isomer of cdATP could be inserted opposite to the dTMP, but the R isomer of cdATP could be inserted opposite to the dCMP. Kinetic analysis revealed that the S isomer of cdATP could be incorporated more efficiently (25.6 μM(-1) min(-1)) than the R isomer (1.13 μM(-1) min(-1)) during DNA synthesis. Previous data showed that the S isomer in DNA blocked DNA synthesis and the exonuclease activity of DNA polymerase and is less efficiently repaired by NER. This indicates that the S isomer has a tendency to accumulate on the genome DNA, and as such, the S isomer of cdATP may be a candidate cytotoxic drug.

SMAD7 and MGMT genotype variants and cancer incidence in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk Study

Purpose: The SMAD7 gene was recently identified to be associated with colorectal cancer risk. Smad7 protein is a known inhibitor of TGF-β signalling pathway which has a prominent role in tumorigenesis. MGMT gene regulates the direct damage reversal repair pathway, preventing DNA damage and potential cancer development. This exploratory study aims to investigate the association between SMAD7 (rs4464148, rs4939827) and MGMT (rs12917, rs2308321) genotype variants, and all-cancer incidence. Methods: Our study population was a sub-cohort of the EPIC-Norfolk study, a prospective cohort of approximately 25,000 men and women aged 40–79. Between recruitment 1993–1997 and follow-up to 2006, 192 incident cases and 1155 non-cases with genotype data were identified. Baseline 7-day food diary and health/lifestyle questionnaire data were analysed. Results: SMAD7 rs4464148 variant genotype was associated with increased cancer incidence [HR = 1.34, 95%CI = 1.00–1.80] but no overall association for SMAD7 rs4939827 or MGMT genotypes. Participants with variant genotypes in both SMAD7 SNPs had a higher cancer incidence compared to those without any (HR = 2.74, 95%CI = 1.10–6.79) ( P = 0.03; P trend = 0.01). Amongst the younger age participants (<mean 62 years), SMAD7 rs4464148 variant genotype group had higher HR of 1.71 (95%CI = 1.03–2.83) compared to the non-variant genotype group. Conclusions: These findings suggest that SMAD7 rs4464148 common variant genotype is associated with cancer incidence but not SMAD7 rs4939827, MGMT rs12917 or MGMT rs2308321. A combination of variants of the SMAD7 SNPs may be associated with increased cancer risk in this study population. However, these findings need to be replicated in larger studies and in studies of specific cancer sites.

Unlocking the Molecular Mechanisms of DNA Repair and Platinum Drug Resistance in Cancer Chemotherapy

This review article is devoted exclusively to DNA repair and acquired resistance to platinum compounds, with an emphasis on research needs and clinical application. It focuses on the role of genetic variants (gene mutations and SNPs) and epigenetic alterations (methylation and acetylation). Four major DNA repair pathways and one enzymecorrection mechanism are presented: Base Excision Repair, Nucleotide Excision Repair, DNA Double-Strand Break Repair, Mismatch Repair and Direct Damage Reversal. It is suggested that one cause of platinum resistance is more accurately described as alterations of DNA repair system rather than activation of DNA repair mechanism, as this cause of resistance is brought about by changes in genetic and epigenetic regulation. Given what is known, research on DNA repair and platinum resistance might best be directed at 1) transcriptional cis-elements (activators/repressors) within promoters of essential DNA repair genes, 2) effects of epigenetic alterations, and 3) connections between gene expression and DNA methylation or protein acetylation. Special attention should be directed at three interrelationships: between the different DNA repair pathways; between DNA methylation and protein acetylation; and between DNA repair pathways and DNA methylation or protein acetylation. Improved clinical outcomes may be achieved by restoring wild type p53 with small molecule drugs, the use of gene demethylation strategies, individual-targeted treatment of BRCA mutation carrier, and combining platinum compounds with molecularly-targeted drugs such as EGFR inhibitors. Keywords: DNA repair, platinum resistance, genetic alteration, methylation, acetylation, epigenetic regulation

DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy

O 6-Alkylguanine-DNA alkyltransferase (AGT) is a crucial target both for the prevention of cancer and for chemotherapy, since it repairs mutagenic lesions in DNA, and it limits the effectiveness of alkylating chemotherapies. AGT catalyzes the unique, single-step, direct damage reversal repair of O 6-alkylguanines by selectively transferring the O 6-alkyl adduct to an internal cysteine residue. Recent crystal structures of human AGT alone and in complex with substrate DNA reveal a two-domain α/β fold and a bound zinc ion. AGT uses its helix-turn-helix motif to bind substrate DNA via the minor groove. The alkylated guanine is then flipped out from the base stack into the AGT active site for repair by covalent transfer of the alkyl adduct to Cys145. An asparagine hinge (Asn137) couples the helix-turn-helix DNA binding and active site motifs. An arginine finger (Arg128) stabilizes the extrahelical DNA conformation. With this newly improved structural understanding of AGT and its interactions with biologically relevant substrates, we can now begin to unravel the role it plays in preserving genetic integrity and discover how it promotes resistance to anticancer therapies.

Structural basis for enzymatic excision of N1-methyladenine and N3-methylcytosine from DNA

N(1)-methyladenine (m(1)A) and N(3)-methylcytosine (m(3)C) are major toxic and mutagenic lesions induced by alkylation in single-stranded DNA. In bacteria and mammals, m(1)A and m(3)C were recently shown to be repaired by AlkB-mediated oxidative demethylation, a direct DNA damage reversal mechanism. No AlkB gene homologues have been identified in Archaea. We report that m(1)A and m(3)C are repaired by the AfAlkA base excision repair glycosylase of Archaeoglobus fulgidus, suggesting a different repair mechanism for these lesions in the third domain of life. In addition, AfAlkA was found to effect a robust excision of 1,N(6)-ethenoadenine. We present a high-resolution crystal structure of AfAlkA, which, together with the characterization of several site-directed mutants, forms a molecular rationalization for the newly discovered base excision activity.

Polymorphisms of DNA repair genes and risk of non-small cell lung cancer

Lung cancer is a leading cause of cancer mortality with an inter-individual difference in susceptibility to the disease. The inheritance of low-efficiency genotypes involved in DNA repair and replication may contribute to the difference in susceptibility. We investigated 44 single nucleotide polymorphisms (SNPs) in 20 DNA repair genes including nucleotide excision repair (NER) genes XPA, ERCC1, ERCC2/XPD, ERCC4/XPF and ERCC5/XPG; base excision repair (BER) genes APE1/APEX, OGG1, MPG, XRCC1, PCNA, POLB, POLiota, LIG3 and EXO1; double-strand break repair (DSB-R) genes XRCC2, XRCC3, XRCC9, NBS1 and ATR; and direct damage reversal (DR) gene MGMT/AGT. The study included 343 non-small cell lung cancer (NSCLC) cases and 413 controls from Norwegian general population. Our results indicate that SNPs in the NER genes ERCC1 (Asn118Asn, 15310G>C, 8902G>T), XPA (-4G>A), ERCC2/XPD (Lys751Gln) and ERCC5/XPD (His46His); the BER genes APE1/APEX (Ile64Val), OGG1 (Ser326Cys), PCNA (1876A>G) and XRCC1 (Arg194Trp, Arg280His, Arg399Gln); and the DSB-R genes ATR (Thr211Met), NBS1 (Glu185Gln), XRCC2 (Arg188His) and XRCC9 (Thr297Ile) modulate NSCLC risk. The level of polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in normal lung tissue from 211 patients was analysed. The variant alleles of XRCC1(Arg280His), XRCC1 (Arg399Gln), ERCC1(G8092T), ERCC5(His46His) and MGMT/AGT(Lys178Arg) were more frequent in patients with PAH-DNA adduct levels lower than the mean whereas the XRCC1(Arg194Trp) variant was more frequent in cases with higher adduct levels than the mean.

Interrogating DNA Repair in Cancer Risk Assessment

DNA is constantly injured by external stress such as exposure to tobacco-smoke constituents, sunlight, or dietary constituents, such as charbroiled meat products, and internal stress, such as free radicals associated with oxygen metabolism. DNA lesions interfere with replication and with

DNA binding and nucleotide flipping by the human DNA repair protein AGT.

O(6)-alkylguanine-DNA alkyltransferase (AGT), or O(6)-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines. AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway. AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials. We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O(6)-methylguanine or crosslinked to the mechanistic inhibitor N(1),O(6)-ethanoxanthosine. The prototypical DNA major groove-binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding. This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2. Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.

Properties of Bacteriophage T4 Proteins Deficient in Replication Repair

An epistasis group of mutations engendering increased sensitivity to diverse DNA-damaging agents was described previously in bacteriophage T4. These mutations are alleles of genes 32 and 41, which, respectively, encode a single-stranded DNA-binding protein (gp32) and the replicative DNA helicase (gp41). The mechanism by which the lethality of DNA damage is mitigated is unknown but seems not to involve the direct reversal of damage, excision repair, conventional recombination repair, or translesion synthesis. Here we explore the hypothesis that the mechanism involves a switch in DNA primer extension from the cognate template to an alternative template, the just-synthesized daughter strand of the other parental strand. The activities of the mutant proteins are reduced about 2-fold (for gp32) or 4-fold (for gp41) in replication complexes catalyzing coordinated synthesis of leading and lagging strands, in binding single-stranded DNA, promoting DNA annealing, and promoting branch migration. In striking contrast, the mutant proteins are strongly impaired in promoting template switching, thus supporting the hypothesis of survival by template switching.

Reversal of DNA alkylation damage by two human dioxygenases.

The Escherichia coli AlkB protein protects against the cytotoxicity of methylating agents by repair of the DNA lesions 1-methyladenine and 3-methylcytosine, which are generated in single-stranded stretches of DNA. AlkB is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase that oxidizes the relevant methyl groups and releases them as formaldehyde. Here, we identify two human AlkB homologs, ABH2 and ABH3, by sequence and fold similarity, functional assays, and complementation of the E. coli alkB mutant phenotype. The levels of their mRNAs do not appear to correlate with cell proliferation but tissue distributions are different. Both enzymes remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides in an alpha-ketoglutarate-dependent reaction, and act by direct damage reversal with the regeneration of the unsubstituted bases. AlkB, ABH2, and ABH3 can also repair 1-ethyladenine residues in DNA with the release of acetaldehyde.

Removal of oxygen free-radical-induced 5',8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells.

Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5' position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5'R or 5'S form of 5',8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex. The R diastereoisomer was more efficiently repaired than the S isomer. No correction of the lesion by direct damage reversal or base excision repair was detected. Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals.

Mitochondrial DNA repair pathways

DNA repair mechanisms are fairly well characterized for nuclear DNA while knowledge regarding the repair mechanisms operable in mitochondria is limited. Several lines of evidence suggest that mitochondria contain DNA repair mechanisms. DNA lesions are removed from mtDNA in cells exposed to various chemicals. Protein activities that process damaged DNA have been detected in mitochondria. As will be discussed, there is evidence for base excision repair (BER), direct damage reversal, mismatch repair, and recombinational repair mechanisms in mitochondria, while nucleotide excision repair (NER), as we know it from nuclear repair, is not present.