The role of O⁶-alkylguanine-DNA alkyltransferase in protecting Rat4 cells against the mutagenic effects of O⁶-substituted guanine residues incorporated in codon 12 of the H-ras gene

Abstract

The role of rat O6-alkylguanine-DNA alkyltransferase (AGT) in modulating mutagenesis by O6-substituted guanines in the rat H-ras gene was examined. Rat4 cells were transfected with vectors carrying O6-methyl-, O6-ethyl- or O6-benzylguanine residues in place of the normal guanines at either the first, second, or both the first and second positions in codon 12 (GGA) of the H-ras coding sequence. The percentage of transformed colonies was determined for cells grown in normal medium or in medium containing O6-benzylguanine to completely deplete AGT. In parallel experiments with O6-methylguanine-containing vectors, the percentage of cellular DNA harboring codon 12 mutations was determined for normal cells and cells lacking AGT. A reasonable correspondence was observed between the percentage of mutated DNA and the percentage of transformed colonies produced in both types of cells. The results indicate that the contribution of AGT to the repair of O6-substituted guanine damage decreases as the O6 substituent is changed from methyl > ethyl > benzyl. Additionally, cellular AGT appears to repair an O6-methylguanine more readily at the first position of codon 12 than the second position. However, other repair mechanisms in these mammalian cells appear to play a major role in correcting low levels of O6-substituted guanine damage including O6-methylguanine damage.

Analysis of tissue-specific lacZ mutations induced by N- nitrosodibenzylamine in transgenic mice

“…The primary mechanism for the aetiology of these G:C→A:T transitions is likely deamination of 5-meC, leading to formation of thymine, since a significant number of mutations were detected at 5Ј-CpG-3Ј sites. The recovery of this mutation at non-CpG sites after NDBzA treatment may be caused by O 6 -benzylG, which also produce G→A transitions (35,36). In fact, a significant number of G:C→A:T transitions (10/18 and 5/12 respectively) were detected at 5Ј-GG-3Ј sites, a characteristic not seen in the spontaneous spectrum (18,30).…”

“…It has been suggested that O 6 -benzylG may exert its mutagenic potential through semi-targeted as well as targeted pathways (35). O 6 -BenzylG may induce transitions and/or transversions depending on the surrounding sequence content (35,36). When incorporated at the second guanine position in a 5Ј-GGA-3Ј sequence (H-ras codon 12), O 6 -benzylG induced G→A, G→T and G→C mutations, but induced exclusively G→A mutations when 2242 incorporated at the first guanine position.…”

“…This tendency was also observed in the 90 day spectrum, albeit less significantly. Nevertheless, the rapid and efficient depletion of O 6 -alkylguanine alkyltransferase by benzylated adducts (36,37) could also play a role in the type of mutation generated, likely through enhancing endogenous mutagenic intermediates, which are normally repaired by this enzyme. In addition, the oxygen free radicals generated during NDBzA metabolism may also play a role by inhibiting cellular DNA repair (38) and by inducing mutations such as G→T transversion (39).…”

Lethality to leukemia cell lines of DNA interstrand cross-links generated by Cloretazine derived alkylating species

“…In contrast, O 6 -chloroethylguanine undergoes a spontaneous rearrangement to produce the highly lethal G-C ethane cross-link which cannot be repaired by AGT [11]. Furthermore, the initial lesion generated by 90CE is likely to be a poorer substrate for AGT than the O 6 -methylguanine lesion [30], which has an in vivo t 1/2 in rat liver of 47 min [26]. Thus, for a cell to survive, the AGT level must be large enough to produce a repair rate that clears the G-C ethane cross-link precursor lesions before their transition to a lethal number of cross-links (<10).…”

Mutagenesis by O6-Methyl-, O6-Ethyl-, and O6-Benzylguanine and O4-Methylthymine in Human Cells: Effects of O6-Alkylguanine-DNA Alkyltransferase and Mismatch Repair

“…Aspects of the mutagenicity and repair of these types of modified bases have been studied previously in mammalian cells using the site-specific mutagenesis approach (1)(2)(3)(4)(5)(6)(7)(8)(9). We have used this approach here to examine mutagenesis and repair of O 6 -methyl (m 6 G)-, O 6 -ethyl (e 6 G)-, and O 6 -benzylguanine (b 6 G), as well O 4 -methylthymine (m 4 T) in human cells.…”