Release of N2,3-ethenoguanine from chloroacetaldehyde-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

Abstract

The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which form N2,3-ethenoguanine in DNA. N2,3-Ethenoguanine is known to cause G --A transitions during DNA replication inEscherichia coli, and its formation may be a carcinogenic event in higher organisms. To investigate the repair ofN2,3-ethenoguanine, we have prepared an N2,3-etheno[14Clguanine-containing DNA substrate by nick-translating DNA with [14CldGTP and modifying the product with chloroacetaldehyde. E. coli 3-methyladenine DNA glycosylase II, purified from cells which carry the plasmid pYN1000, releases N2,3-ethenoguanine from chloroacetaldehyde-modified DNA in a protein-and time-dependent manner. This finding widens the known substrate specificity of glycosylase II to include a modified base which may be associated with the carcinogenic process. Similar enzymatic activity in eukaryotic cells might protect them from exposure to metabolites of vinyl chloride. gated the activity of this enzyme towards an EG-containing DNA substrate.Both glycosylase II and 06-alkylguanine-DNA alkyltransferase are synthesized by E. coli in increased amounts as part of the adaptive response to methylating carcinogens (17). 06-alkylguanine-DNA alkyltransferase removes alkyl groups from the Q6 position of guanine and from phosphotriesters, thereby restoring the original structure. Glycosylase II acts in a different way, releasing modified bases from the DNA and leaving apurinic sites which are subject to further repair.Glycosylase II releases 3-and 7-alkylated purines as well as 02-methylcytosine and 02-methylthymine from DNA (17). The recent finding that N2,3-ethanoguanine is also released extends the specificity of this enzyme to DNA bases that bear exocyclic groups. The studies reported here show that the enzyme has activity towards a base that has been associated with vinyl chloride carcinogenicity.The industrial chemical vinyl chloride is carcinogenic in animal tests and is associated with an increased incidence of hepatic angiosarcomas in exposed workers (1). Vinyl chloride is metabolized by the cytochrome P450 system in the liver to the unstable electrophile chloroethylene oxide, which rearranges spontaneously to the more stable alkylating agent chloroacetaldehyde (CAA) (2, 3). Chloroethylene oxide and CAA react with DNA to form a variety of adducts, many of which have been identified in vivo (4-10). The question has arisen, therefore, as to which DNA modification or modifications may be responsible for initiating the carcinogenic process.The modified base that is found in the greatest quantity in livers of animals exposed to vinyl chloride is 7-(2-oxoethyl)-guanine (oeG) (4, 6, 7). This base does not cause misincorporation when it is present in a DNA template strand (11), but it does form apurinic sites, which could be mutagenic or carcinogenic. On the other hand, N2,3-ethenoguanine (EG) has been shown to mispair in in vitro transcription systems (12, 13) and causes G --A transitions in Escherichia coli (14).These results...

DNA glycosylases in the base excision repair of DNA

“…Not only are these substrates different in the sense that they represent pyrimidines and purines but, in addition, the methyl group of 7-meG protrudes into the major groove, whereas the other alkyl groups protrude into the minor groove. AlkA also removes alkylation products of the bifunctional alkylating agent chloroethylnitrosourea, such as 7-hydroxyethylguanine, 7-chloroethylguanine and some minor alkylation products [137], as well as the cyclic etheno adducts 1,N'-ethenoadenine, 1,N#-ethenoguanine and 3,N%-ethenocytosine induced by vinyl chloride or chloroacetaldehyde [138,139]. Adducts formed by sulphur mustard [140] and nitrogen mustards [141] are also recognized by AlkA.…”

Evidence for the presence of kinetin in DNA and cell extracts

“…First, it can induce the synthesis of repair enzymes which remove modified bases from DNA [21][22][23] and/or are involved in protection against oxygen stress [22]. Second, its complex with copper(II) initiates superoxide dismutase, in that the kinetin-Cu(II) complex catalyses 0 2 dismutation very efficiently at physiological pH, with a turnover of 2.7x 10 9 mol per s [24][25][26].…”

Repair of exocyclic DNA adducts: rings of complexity

“…Ludlum's group (71) first reported release of N 2 ,3-eG from CAA-treated DNA by the purified E. coli AlkA protein (3mA-DNA glycosylase II) and estimated that this novel activity is only 1/20th of the 3mA activity. A low-level release of this adduct was also found by Singer's laboratory in cell-free extracts of both HeLa cells and an E. coli strain expressing hANPG.…”

“…(78) The same enzyme also releases the closely related N 2 ,3-eG, (71) although the relative efficiency of these two activities has not been compared. Recently, EA in DNA was shown to be excised by both hANPG and AlkA, but with significantly lower efficiencies as compared with eA.…”