Repair Of Alkylation Damage Research Articles

Inhibition of human DNA alkylation damage repair enzyme ALKBH2 by HIV protease inhibitor ritonavir

The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA and is overexpressed in several cancers. However, there are no known inhibitors available for this crucial DNA repair enzyme. The aim of this study was to examine whether the first-generation HIV protease inhibitors having strong anti-cancer activity can be repurposed as inhibitors of ALKBH2. We selected four such inhibitors and performed in vitro binding analysis against ALKBH2 based on alterations of its intrinsic tryptophan fluorescence and differential scanning fluorimetry. The effect of these HIV protease inhibitors on the DNA repair activity of ALKBH2 was also evaluated. Interestingly, we observed that one of the inhibitors, ritonavir, could inhibit ALKBH2-mediated DNA repair significantly via competitive inhibition and sensitized cancer cells to alkylating agent methylmethane sulfonate (MMS). This work may provide new insights into the possibilities of utilizing HIV protease inhibitor ritonavir as a DNA repair antagonist.

How to sensitize glioblastomas to temozolomide chemotherapy: a gap-centered view.

Temozolomide (TMZ) is a methylating agent used as the first-line drug in the chemotherapy of glioblastomas. However, cancer cells eventually acquire resistance, necessitating the development of TMZ-potentiating therapy agents. TMZ induces several DNA base adducts, including O 6 -meG, 3-meA, and 7-meG. TMZ cytotoxicity stems from the ability of these adducts to directly (3-meA) or indirectly (O 6 -meG) impair DNA replication. Although TMZ toxicity is generally attributed to O 6 -meG, other alkylated bases can be similarly important depending on the status of various DNA repair pathways of the treated cells. In this mini-review we emphasize the necessity to distinguish TMZ-sensitive glioblastomas, which do not express methylguanine-DNA methyltransferase (MGMT) and are killed by the futile cycle of mismatch repair (MMR) of the O 6 -meG/T pairs, vs. TMZ-resistant MGMT-positive or MMR-negative glioblastomas, which are selected in the course of the treatment and are killed only at higher TMZ doses by the replication-blocking 3-meA. These two types of cells can be TMZ-sensitized by inhibiting different DNA repair pathways. However, in both cases, the toxic intermediates appear to be ssDNA gaps, a vulnerability also seen in BRCA-deficient cancers. PARP inhibitors (PARPi), which were initially developed to treat BRCA1/2-deficient cancers by synthetic lethality, were re-purposed in clinical trials to potentiate the effects of TMZ. We discuss how the recent advances in our understanding of the genetic determinants of TMZ toxicity might lead to new approaches for the treatment of glioblastomas by inhibiting PARP1 and other enzymes involved in the repair of alkylation damage (e.g., APE1).

Base Excision DNA Repair in Plants: Arabidopsis and Beyond.

Base excision DNA repair (BER) is a key pathway safeguarding the genome of all living organisms from damage caused by both intrinsic and environmental factors. Most present knowledge about BER comes from studies of human cells, E. coli, and yeast. Plants may be under an even heavier DNA damage threat from abiotic stress, reactive oxygen species leaking from the photosynthetic system, and reactive secondary metabolites. In general, BER in plant species is similar to that in humans and model organisms, but several important details are specific to plants. Here, we review the current state of knowledge about BER in plants, with special attention paid to its unique features, such as the existence of active epigenetic demethylation based on the BER machinery, the unexplained diversity of alkylation damage repair enzymes, and the differences in the processing of abasic sites that appear either spontaneously or are generated as BER intermediates. Understanding the biochemistry of plant DNA repair, especially in species other than the Arabidopsis model, is important for future efforts to develop new crop varieties.

Integrative modeling of the ASCC2:K63Ub2 complex to better understand DNA alkylation damage repair

Integrative modeling of the ASCC2:K63Ub2 complex to better understand DNA alkylation damage repair

Use of High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) to Quantify Modified Nucleosides.

Physiological and chemically induced modifications to nucleosides are common in both DNA and RNA. Physiological forms of these modifications play critical roles in gene expression, yet aberrant marks, if left unrepaired, may be associated with increased genome instability. Due to the low prevalence of these marks in most samples of interest, a highly sensitive method is needed for their detection and quantitation. High-performance liquid chromatography, coupled to mass spectrometry (HPLC-MS), provides this high degree of sensitivity while also being adaptable to nearly any modified nucleoside of interest and still maintaining exquisite specificity. In this chapter, we demonstrate how to use HPLC-MS to analyze the catalytic activity of a nucleic acid demethylase, to quantify the prevalence of N6-methyladenosine from RNA, and to determine the kinetics of alkylation damage repair.

Versatile cell-based assay for measuring DNA alkylation damage and its repair

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.

Mitochondrial DNA Abundance is Maintained by APE1 in Liver of Mice Treated with AOM

Background The liver is the body's largest internal organ which performs many metabolic processes, including detoxification of several classes of carcinogens. Because of the numerous critical functions performed by the liver, degenerative liver diseases such as hepatitis, cirrhosis, and liver cancer are especially life-threatening. Previous research indicates, that in liver cancer, mitochondrial function is altered. Base Excision Repair (BER) is the primary mechanism for the repair of alkylation and oxidative DNA damage in mitochondria. Our research is focusing on the study of the protein apurinic/apyrimidinic endonuclease 1 (APE 1). The alkylating agent azoxymethane (AOM) is a carcinogen widely used for the induction of colorectal cancer in rodents. AOM is bioactivated in the liver, thus this organ could represent a primary target of AOM action and a model for liver carcinogenesis. Objective To assess the effect of AOM in mtDNA abundance in liver tissue from mice deficient in the main abasic site endonuclease APE1. Hypothesis Mitochondrial DNA (mtDNA) abundance will be altered in liver tissue after AOM treatment. Methods Two strains of mice were analyzed, a Wild Type (WT) strain and another carrying a null mutation of one allele of the DNA repair gene APE1 (Apex1+/-). Animals were exposed to 4 doses of AOM (10mg/kg body weight once a week for 4 weeks) and liver tissue was harvested 6 months after the first AOM treatment. MtDNA abundance was analyzed by real-time PCR. Results We observed a 30% decreased in mtDNA abundance in liver tissue from Apex1+/- mice chronically treated with AOM when compared to its control, while in WT animals mtDNA abundance was unchanged. Conclusion Our results indicate that chronic AOM treatment in DNA repair deficient mice results in decreased mtDNA abundance in liver tissue. Further research will focus on the consequences of loss of mtDNA abundance in mitochondrial function.

DNA alkylation lesion repair: outcomes and implications in cancer chemotherapy.

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, represent a major form of DNA damage in cells. The repair of alkylation damage is critical in all cells because such damage is cytotoxic and potentially mutagenic. Alkylation chemotherapy is a major therapeutic modality for many tumors, underscoring the importance of the repair pathways in cancer cells. Several different pathways exist for alkylation repair, including base excision and nucleotide excision repair, direct reversal by methyl-guanine methyltransferase (MGMT), and dealkylation by the AlkB homolog (ALKBH) protein family. However, maintaining a proper balance between these pathways is crucial for the favorable response of an organism to alkylating agents. Here, we summarize the progress in the field of DNA alkylation lesion repair and describe the implications for cancer chemotherapy.

The interaction of DNA repair factors ASCC2 and ASCC3 is affected by somatic cancer mutations

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2–ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2–ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2–ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.

Expansion of base excision repair compensates for a lack of DNA repair by oxidative dealkylation in budding yeast

The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli. However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.

Complex genetic interactions between DNA polymerase β and the NHEJ ligase.

Mammalian cells possess multiple pathways for repairing various types of DNA damage. Although the molecular mechanisms of each DNA repair pathway have been analyzed by biochemical analysis and cell biological analysis, interplay between different pathways has not been fully elucidated. In this study, using human Nalm-6-mutant cell lines, we analyzed the relationship between the base excision repair factor DNA polymerase β (POLβ) and DNA ligase IV (LIG4), which is essential for DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). We found that cells lacking both POLβ and LIG4 grew significantly more slowly than either single mutant, indicating cooperative functions of the two proteins in normal cell growth. To further investigate the genetic interaction between POLβ and LIG4, we examined DNA damage sensitivity of the mutant cell lines. Our results suggested that NHEJ acts as a backup pathway for repairing alkylation damage (when converted into DSBs) in the absence of POLβ. Surprisingly, despite the critical role of POLβ in alkylation damage repair, cells lacking POLβ exhibited increased resistance to camptothecin (a topoisomerase I inhibitor that induces DNA single-strand breaks), irrespective of the presence or absence of LIG4. A LIG4-independent increased resistance associated with POLβ loss was also observed with ionizing radiation; however, cells lacking both POLβ and LIG4 were more radiosensitive than either single mutant. Taken together, our findings provide novel insight into the complex interplay between different DNA repair pathways.

The DNA Alkylguanine DNA Alkyltransferase-2 (AGT-2) Of Caenorhabditis Elegans Is Involved In Meiosis And Early Development Under Physiological Conditions

DNA alkylguanine DNA alkyltransferases (AGTs) are evolutionary conserved proteins that repair alkylation damage in DNA, counteracting the effects of agents inducing such lesions. Over the last years AGTs have raised considerable interest for both the peculiarity of their molecular mechanism and their relevance in cancer biology. AGT knock out mice show increased tumour incidence in response to alkylating agents, and over-expression of the human AGT protein in cancer cells is frequently associated with resistance to alkylating chemotherapy. While all data available point to a function of AGT proteins in the cell response to alkylation lesions, we report for the first time that one of the two AGT paralogs of the model organism C. elegans, called AGT-2, also plays unexpected roles in meiosis and early development under physiological conditions. Our data suggest a role for AGT-2 in conversion of homologous recombination intermediates into post-strand exchange products in meiosis, and show that agt-2 gene down-regulation, or treatment of animals with an AGT inhibitor results in increased number of germ cells that are incompatible with producing viable offspring and are eliminated by apoptosis. These results suggest possible functions for AGTs in cell processes distinct from repair of alkylating damage.

Heteroexpression of Mycobacterium leprae hypothetical protein ML0190 provides protection against DNA-alkylating agent methyl methanesulfonate

Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage.

The DNA damage response is developmentally regulated in the African trypanosome

Genomes are affected by a wide range of damage, which has resulted in the evolution of a number of widely conserved DNA repair pathways. Most of these repair reactions have been described in the African trypanosome Trypanosoma brucei, which is a genetically tractable eukaryotic microbe and important human and animal parasite, but little work has considered how the DNA damage response operates throughout the T. brucei life cycle. Using quantitative PCR we have assessed damage induction and repair in both the nuclear and mitochondrial genomes of the parasite. We show differing kinetics of repair for three forms of DNA damage, and dramatic differences in repair between replicative life cycle forms found in the testse fly midgut and the mammal. We find that mammal-infective T. brucei cells repair oxidative and crosslink-induced DNA damage more efficiently than tsetse-infective cells and, moreover, very distinct patterns of induction and repair of DNA alkylating damage in the two life cycle forms. We also reveal robust repair of DNA lesions in the highly unusual T. brucei mitochondrial genome (the kinetoplast). By examining mutants we show that nuclear alkylation damage is repaired by the concerted action of two repair pathways, and that Rad51 acts in kinetoplast repair. Finally, we correlate repair with cell cycle arrest and cell growth, revealing that induced DNA damage has strikingly differing effects on the two life cycle stages, with distinct timing of alkylation-induced cell cycle arrest and higher levels of damage induced death in mammal-infective cells. Our data reveal that T. brucei regulates the DNA damage response during its life cycle, a capacity that may be shared by many microbial pathogens that exist in variant environments during growth and transmission.

RNA ligase-like domain in activating signal cointegrator 1 complex subunit 1 (ASCC1) regulates ASCC complex function during alkylation damage

Multiple DNA damage response (DDR) pathways have evolved to sense the presence of damage and recruit the proper repair factors. We recently reported a signaling pathway induced upon alkylation damage to recruit the AlkB homolog 3, α-ketoglutarate-dependent dioxygenase (ALKBH3)-activating signal cointegrator 1 complex subunit 3 (ASCC3) dealkylase-helicase repair complex. As in other DDR pathways, the recruitment of these repair factors is mediated through a ubiquitin-dependent mechanism. However, the machinery that coordinates the proper assembly of this repair complex and controls its recruitment is still poorly defined. Here, we demonstrate that the ASCC1 accessory subunit is important for the regulation of ASCC complex function. ASCC1 interacts with the ASCC complex through the ASCC3 helicase subunit. We find that ASCC1 is present at nuclear speckle foci prior to damage, but leaves the foci in response to alkylation. Strikingly, ASCC1 loss significantly increases ASCC3 foci formation during alkylation damage, yet most of these foci lack ASCC2. These results suggest that ASCC1 coordinates the proper recruitment of the ASCC complex during alkylation, a function that appears to depend on a putative RNA-binding motif near the ASCC1 C terminus. Consistent with its role in alkylation damage signaling and repair, ASCC1 knockout through a CRISPR/Cas9 approach results in alkylation damage sensitivity in a manner epistatic with ASCC3. Together, our results identify a critical regulator of the ALKBH3-ASCC alkylation damage signaling pathway and suggest a potential role for RNA-interacting domains in the alkylation damage response.

Escherichia coli AlkB and single-stranded DNA binding protein SSB interaction explored by Molecular Dynamics Simulation

Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia Coli (E.coli) DNA repair enzyme AlkB removes methyl adducts including 1-methyladenine and 3-methylcytosine present in DNA by oxidative demethylation from single-stranded DNA (ssDNA). E. coli single-stranded DNA binding protein (SSB) selectively binds ssDNA in a sequence-independent manner. We have recently shown that AlkB can repair methyl adduct present in SSB-coated ssDNA. In this study, we aimed to elucidate details of AlkB-mediated DNA repair of SSB-bound DNA substrate. Therefore, we generated a structural model of AlkB-SSB-ssDNA and using Molecular Dynamics simulation analysis we show that flexibility of SSB-bound DNA allows AlkB to bind in multiple ways. Our docking analysis of AlkB-SSB-ssDNA structure revealed that the Cyt109 base is present in the hydrophobic cavity of AlkB active site pocket. The characterization of AlkB-SSB interaction pattern would likely to help in understanding the mode of alkylated DNA adduct recognition by AlkB.

DNA repair activity of Fe(II)/2OG-dependent dioxygenases affected by low iron level in Saccharomyces cerevisiae.

Iron deprivation induces transcription of genes required for iron uptake, and transcription factor Aft1 and Aft2 mediate this by regulating transcriptional program in Saccharomyces cerevisiae. Iron-dependent Fe(II) and 2-oxoglutarate-dependent dioxygenase family proteins are involved in various cellular pathways including DNA alkylation damage repair. Whether Aft1/Aft2 are required for DNA alkylation repair is currently unknown. In this report, we have analyzed DNA alkylation repair under iron-deprived condition. Saccharomyces cerevisiae Tpa1 is a member of Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that deletion of AFT1 and AFT2 genes affects Tpa1 function resulting in sensitivity to alkylating agent methyl methane sulfonate (MMS). Deletion of AFT1 and AFT2 along with base excision repair pathway DNA glycosylase MAG1 renders the aft1Δaft2Δmag1Δ mutant highly sensitive to MMS. We have further studied effect of iron depletion by replacing S. cerevisiae Tpa1 with Escherichia coli AlkB and human AlkBH3. We observed that the activity of AlkB and AlkBH3 is also diminished similarly when present in aft1Δaft2Δ background as evident by sensitivity to MMS.

Escherichia coli single-stranded DNA binding protein SSB promotes AlkB-mediated DNA dealkylation repair

Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia coli (E.coli) protein AlkB removes various alkyl DNA adducts including N1-methyladenine (N1meA) and N3-methylcytosine (N3meC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes N1meA and N3meC from single-stranded DNA (ssDNA). It can also remove N1meA and N3meC from double-stranded DNA by base-flipping. Notably, ssDNA produced during DNA replication and recombination, remains bound to E. coli single-stranded DNA binding protein SSB and it is not known whether AlkB can repair methyl adduct present in SSB-coated DNA. Here we have studied AlkB-mediated DNA repair using SSB-bound DNA as substrate. In vitro repair reaction revealed that AlkB could efficiently remove N3meC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N3meC-containing oligonuleotides were used as the substrate, efficiency of AlkB catalyzed reaction was abated compared to SSB-bound DNA substrate of identical length. Truncated SSB containing only the DNA binding domain could also support the stimulation of AlkB activity, suggesting the importance of SSB-DNA interaction for AlkB function. Using 70-mer oligonucleotide containing single N3meC we demonstrate that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts.

Nucleosomes Regulate Base Excision Repair in Chromatin.

Chromatin is a significant barrier to many DNA damage response (DDR) factors, such as DNA repair enzymes, that process DNA lesions to reduce mutations and prevent cell death; yet, paradoxically, chromatin also has a critical role in many signaling pathways that regulate the DDR. The primary level of DNA packaging in chromatin is the nucleosome core particle (NCP), consisting of DNA wrapped around an octamer of the core histones H2A, H2B, H3 and H4. Here, we review recent studies characterizing how the packaging of DNA into nucleosomes modulates the activity of the base excision repair (BER) pathway and dictates BER subpathway choice. We also review new evidence indicating that the histone amino-terminal tails coordinately regulate multiple DDR pathways during the repair of alkylation damage in the budding yeast Saccharomyces cerevisiae.

A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair.

SummaryDNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signaling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks1–3. Understanding the upstream signaling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation4–6. Here, we demonstrate that human cells have a previously unrecognized signaling mechanism for sensing damage induced by alkylation. We find that the ASCC alkylation repair complex7 relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE domain of ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RNF113A as the E3 ligase responsible for upstream ubiquitin signaling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy (TTD), which harbor a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a heretofore unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked TTD.