Repair of alkylated DNA: Recent advances
Department of Biomedical Sciences , Florida State University, Tallahassee, Florida, United States DNA Repair
(Impact Factor: 3.11).
05/2007; 6(4):429-42. DOI: 10.1016/j.dnarep.2006.10.005
Cytotoxic and mutagenic methylated bases in DNA can be generated by endogenous and environmental alkylating agents. Such damaged bases are removed by three distinct strategies. The abundant toxic lesion 3-methyladenine (3-alkyladenine) is excised by a specific DNA glycosylase that initiates a base excision-repair process. The toxic lesions 1-methyladenine and 3-methylcytosine are corrected by oxidative DNA demethylation catalyzed by DNA dioxygenases. These enzymes release the methyl moiety as formaldehyde, directly reversing the base damage. The third strategy involves the mutagenic and cytotoxic lesion O(6)-methylguanine which is also repaired by direct reversal but uses a different mechanism. Here, the methyl group is transferred from the lesion to a specific cysteine residue within the methyltransferase itself. In this review, we briefly describe endogenous alkylating agents and the extensively investigated DNA repair enzymes, mammalian 3-methyladenine-DNA glycosylase and O(6)-methylguanine-DNA methyltransferase. We provide a more detailed description of the structures and biochemical properties of the recently discovered DNA dioxygenases.
Available from: sciencedirect.com
- "The major enzymes repairing the methylated lesions such as 1-methyladenine (1meA) and 3-methylcytosine (3meC) are the AlkB proteins. Like the JmjC KDMs and TET proteins, AlkB belongs to the Fe(II)-and a-KG-dependent dioxygenases (Falnes et al., 2002) and includes nine distinct genes in human cells (AlkB homolog ALKBH1 to ALKBH8 and FTO) (Sedgwick et al., 2007). The function in repairing DNA alkylation lesion has been demonstrated biochemically in vitro and supported by the genetic analysis of mutant mice for mammalian ALKBH2 and ALKBH3 (Aas et al., 2003; Dango et al., 2011; Duncan et al., 2002; Lee et al., 2005; Ringvoll et al., 2006). "
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ABSTRACT: Chemotherapy of a combination of DNA alkylating agents, procarbazine and lomustine (CCNU), and a microtubule poison, vincristine, offers a significant benefit to a subset of glioma patients. The benefit of this regimen, known as PCV, was recently linked to IDH mutation that occurs frequently in glioma and produces D-2-hydroxyglutarate (D-2-HG), a competitive inhibitor of α-ketoglutarate (α-KG). We report here that D-2-HG inhibits the α-KG-dependent alkB homolog (ALKBH) DNA repair enzymes. Cells expressing mutant IDH display reduced repair kinetics, accumulate more DNA damages, and are sensitized to alkylating agents. The observed sensitization to alkylating agents requires the catalytic activity of mutant IDH to produce D-2-HG and can be reversed by the deletion of mutant IDH allele or overexpression of ALKBH2 or AKLBH3. Our results suggest that impairment of DNA repair may contribute to tumorigenesis driven by IDH mutations and that alkylating agents may merit exploration for treating IDH-mutated cancer patients. Wang et al. demonstrate that D-2-HG produced by mutant IDH inhibits alkylated DNA repair enzymes, leading to DNA damage and sensitizing IDH mutant cells to alkylating agents. These results suggest that impairment of DNA repair may contribute to tumorigenesis driven by IDH mutations and that alkylating agents should be explored as a therapeutic option for IDH-mutated cancer patients.
- "AlkB protein in the presence of non-heme Fe(II) and cofactors, 2-oxoglutarate (2OG) and oxygen (O 2 ) removes alkyl adducts recovering native DNA structure. It acts more effectively in single stranded (ss) DNA and RNA than double stranded (ds) DNA   . "
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ABSTRACT: An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH+ strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg+ reversion induction after λ > 320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg+ revertants in AB1157 alkB+hemH–/pMW1 strain was 40 and 26% reduced comparing to the alkB+ hemH– and alkB+ hemH+/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ > 320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg+ reversion was observed in irradiated and MMS-treated hemH– alkB– cells comparing to the hemH+ alkB– strain. Also, the level of Arg+ revertants in the irradiated and MMS treated hemH– alkB– mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH– intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.
Available from: Yu Zhao
- "Alkylation is a major mechanism by which nucleic acids are modified, and repair of these adducts is critical since they are mutagenic and may cause DNA breaks (Sedgwick et al, 2007; Fu et al, 2012). Alkylation damage is repaired by at least three different pathways, including base-excision repair (BER), suicidal methyltransferases such as methyl-guanine methyltransferase (MGMT), and direct methylation/ alkylation reversal by the AlkB family of demethylases (Drablos et al, 2004; Sedgwick et al, 2007; Fu et al, 2012). Originally discovered in Escherichia coli, the AlkB protein catalyzes oxidative demethylation of cytotoxic adducts of DNA bases alkylated at the N1 position of purines and N3 position of pyrimidines (Falnes et al, 2002; Trewick et al, 2002). "
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ABSTRACT: Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.
© 2015 The Authors.
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