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Recent Progress on the Ada Response for Inducible Repair of DNA Alkylation Damage

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Recent Progress on the Ada Response for Inducible Repair of DNA Alkylation Damage Oncogene (2002) 21, 8886 – 8894 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Recent progress on the Ada response for inducible repair of DNA alkylation damage Barbara Sedgwick1 and Tomas Lindahl*,1 1Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK Oncogene (2002) 21, 8886 – 8894. doi:10.1038/sj.onc. Methylating agents 1205998 Methylating agents can alkylate DNA at many sites producing a wide variety of base lesions (Figure 1) and Keywords: DNA repair; O6-methylguanine; DNA phosphotriesters. The relative proportions of the glycosylases different base lesions depend on the nature of the alkylating agent, its reaction mechanism and the secondary structure of the DNA target. Of the commonly used laboratory alkylating agents, methyl Introduction methanesulphonate (MMS), dimethylsulphate (DMS) and methyl iodide (MeI) react via a SN2 mechanism Methylating agents comprise a major class of DNA and methylate DNA almost exclusively at nitrogen damaging compounds that occur both endogenously moieties in the purine and pyrimidine rings. and in the environment. They are extremely cytotoxic In contrast, N-methyl-N’-nitro-N-nitrosoguanidine and frequently also mutagenic, and are employed for (MNNG) and N-methylnitrosourea (MNU), which chemotherapy of certain cancers. All organisms have are SN1 agents, alkylate both nitrogens and oxygens multiple DNA repair strategies to counteract the effects in the DNA bases and also oxygens in the sugar- of DNA alkylation. To defend against fluctuating phosphate backbone. The major lesions generated in levels of environmental alkylating agents, many double-stranded DNA are 7-methylguanine (7-meG), bacteria mount an inducible response that enhances 3-methyladenine (3-meA) and O6-methylguanine (O6- cellular resistance to these same agents. This adaptive meG). In single-stranded DNA, SN2 agents also response has been most extensively studied in E. coli,in efficiently induce formation of 1-methyladenine (1- which induced alkylation resistance results from meA) and 3-methylcytosine (3-meC) (Bodell and increased expression of four genes, ada, alkB, alkA Singer, 1979). These lesions are less readily formed in and aidB. The Ada, AlkA and AlkB proteins protect duplex DNA because the modification sites are against alkylation by repair of methylated bases in involved in base pairing, and are therefore shielded DNA using different mechanisms, whereas AidB may from alkylation. There are also some distinct minor directly destroy certain alkylating agents. These DNA sites of methylation. Several of the base modifications repair activities are conserved from bacteria to man. block DNA replication and are cytotoxic, including 3- The Ada protein also serves as the positive regulator of meA, 3-methylguanine (3-meG), 3-meC and probably ‘the adaptive response to alkylation damage’, which is also 1-meA. O6-meG and O4-methylthymine (O4-meT) more concisely named ‘the Ada response’ by analogy mispair during DNA replication and are therefore with the SOS, SoxRS and OxyR responses. Various mutagenic. It is fortuitous that the most abundantly aspects of the Ada response have been the subject of formed lesion, 7-meG, is relatively innocuous (Singer previous detailed reviews (Friedberg et al., 1995; and Grunberger, 1983). In a different type of DNA Landini and Volkert, 2000; Lindahl et al., 1988; alkylation, environmental mutagens such as 1,2- Samson, 1992; Sedgwick and Vaughan, 1991; Seeberg dimethylhydrazine, tert-butylhydroperoxide and diazo- and Berdal, 1999). Here, we describe the properties of quinones generate methyl radicals that react with the inducible proteins with particular emphasis on guanine residues in DNA to form miscoding 8- recent findings, such as those from genome sequencing, methylguanine adducts (Hix et al., 1995). protein structural studies, analysis of substrate specifi- Conservation of the Ada response in many bacterial cities, and the newly discovered function of the AlkB species suggests the presence of direct alkylating agents protein. in their environment. Good candidate agents are those produced by microorganisms, but others may be formed by chemical reactions. Some Streptomyces sp. release alkylating antibiotics, such as streptozotocin (a derivative of MNU) and azaserine (O-diazoacyl-L- *Correspondence: T Lindahl; E-mail: [email protected] serine), into the soil creating an urgent need for an Repair of alkylated DNA B Sedgwick and T Lindahl 8887 It is of note that ada gene expression also increases in stationary phase to further the defence against alkylation; this enhanced expression is also dependent on RpoS (Taverna and Sedgwick, 1996). The preli- minary studies on the function of AidB need to be extended, including more precise definition of its eukaryotic homologues. Figure 1 Sites of methylation on the DNA bases. Thick arrows Ada, an O6-meG-DNA methyltransferase and indicate major sites of DNA methylation by SN1 agents. The curly arrow indicates an additional site alkylated by methyl radi- chemosensor for the adaptive response cals. Colours are used to indicate which damaged sites are re- paired by particular DNA repair activities: blue by Ada; green The multifunctional Ada protein repairs methylated by AlkA; red by AlkB; black, no known repair activity bases and also regulates the adaptive response (Figure 2). Ada is composed of two major domains that can function independently in DNA repair reactions. The adaptive response in other microorganisms. Further- 19 kDa C-terminal domain (C-Ada19) directly more, certain algae and fungi growing in saline demethylates the mutagenic bases, O6-meG and O4- environments generate MeCl as a product of chloride meT, and transfers the methyl groups on to its Cys-321 detoxification (Sedgwick and Vaughan, 1991). MeCl is residue (Demple et al., 1985). Ada therefore precludes probably the most abundant methylating agent in our mutation induction by this rapid but suicidal action. environment (Crutzen and Andreae, 1990). Chemically, The active site thiol of Cys-321 is buried in the 3-D direct acting alkylating agents may be formed by structure of C-Ada19. The protein was therefore nitrosations, in slightly acidic conditions, of amides, initially proposed to undergo a substantial conforma- amines, amino acids and peptides (Harrison et al., tional change on binding to its substrates to expose this 1999; Sedgwick, 1997; Sedgwick and Vaughan, 1991). active cysteine (Moore et al., 1994). An alternative These reactions could possibly occur in decaying model, however, used a predicted ‘helix – turn – helix – matter, in acidic soils or in putrid water. wing’ motif (HTH) to bind DNA and a nucleotide E. coli cells that are unable to repair O6-meG or O4- flipping mechanism to align the alkylated guanine with meT residues have an increased frequency of sponta- the catalytic cysteine (Vora et al., 1998). The more neous mutagenesis when subjected to starvation/ recent crystal structure of the human O6-meG-DNA stationary conditions. Mutagenic alkylating agents must therefore arise in these cells, but their precise nature is unknown (Rebeck et al., 1989). S-Adeno- sylmethionine (SAM) weakly methylates DNA, but it acts by an SN2 mechanism and would not be expected to be an efficient mutagen. Thus, variation of the cellular SAM levels, over a 100-fold range, had no significant effect on spontaneous mutagenesis in E. coli (Posnick and Samson, 1999b). It is more likely that the major endogenous mutagen is a SN1 compound that alkylates oxygens in DNA. Bacterially catalyzed amine nitrosation increases in anaerobic and resting E. coli cells (Calmels et al., 1987; Kunisaki and Hayashi, 1979). A mutant deficient in this activity was less susceptible to spontaneous mutagenesis, suggesting that enzymatic nitrosation of amines or amides may be a source of endogenous mutagens (Harrison et al., 1999; Sedgwick, 1997; Taverna and Sedgwick, 1996). AidB protein shows some homology to the mammalian isovaleryl-coenzyme A dehydrogenases, and has been proposed to detoxify nitrosoguanidines (nitrosated amides) or their reaction intermediates (Landini et al., 1994). Expression of the aidB gene increases in anaerobic cells and is dependent on RpoS, the Figure 2 Diagrammatic representation of the E. coli Ada re- alternative sigma factor of RNA polymerase that sponse. The Ada protein is activated as a positive regulator by controls expression of many genes in stationary phase methylation of its Cys-38 residue in the amino-terminal half of (Volkert et al., 1994). A question of interest is whether the protein. This activation occurs by repair of methylphospho- triesters (PTE) in DNA or, less efficiently, by direct protein elevation of AidB activity occurs in the same methylation. The activated Ada protein induces expression of sev- conditions as spontaneous mutagenesis and is required eral genes resulting in increased DNA repair and probable to destroy endogenously generated nitroso-compounds. destruction of certain alkylating agents Oncogene Repair of alkylated DNA B Sedgwick and T Lindahl 8888 methyltransferase (AGT) also implicates a HTH motif Self-methylation at Cys-38 converts Ada into a in DNA binding and an ‘arginine finger’ to extrude the transcriptional activator and dramatically increases its O6-alkG nucleotide from the duplex DNA. The sequence specific binding to the promoters of the ada- extrahelical base can then reach the recessed AGT alkB operon and the alkA and aidB genes (Landini and active site
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