The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

Abstract

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O(6)-carboxymethylguanine (O(6)-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O(6)-CMG is not a substrate for the human version of the DNA damage reversal protein O(6)-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O(6)-alkylguanine lesions by removing alkyl groups from the O(6)-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O(6)-methylguanine (O(6)-MeG) or O(6)-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O(6)-alkyl group and its transfer to the active-site cysteine residue of MGMT. O(6)-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O(6)-CMG is a potential causative agent.

Figure 1.

Inactivation of MGMT (closed symbols) and Ogt (open symbols) by pre-incubation with increasing concentrations of ODN (5′-GCCATGG*CTAGTA) containing O⁶-MeG (squares) or O⁶-CMG (circles) but not G (triangles) for 1 h; values shown are means ± SD. of triplicate determinations. See Materials and Methods for details.

Figure 2.

Incubation of duplex ODNs containing O⁶-MeG (squares) or O⁶-CMG (triangles) with MGMT decreases the binding of Atl1 (A) and anti-CMG antibody (B). No binding to control (G-containing) ODN (diamonds) was seen. To prepare the substrates, ODNs (5′-GCCATGG*CTAGTA, where G* is G, O⁶-MeG or O⁶-CMG) were annealed to complementary ODN containing 5'-terminal biotin and then bound to streptavidin-coated microtitre plates. After incubation with MGMT, binding of Atl1 or anti-O⁶-CMG to the ODN was monitored, as described in Materials and Methods.

Figure 3.

Pre-incubation with MGMT of duplex ODN containing O⁶-MeG and O⁶-CMG enables cleavage by (A) BsaJ1 (in duplexed 5′-GCCATGG*CTAGTA) and (B) PstI (in duplexed 5′-GAACTG*CAGCTCCGTGCTGGCCC). Lanes 1-4, 5-8 and 9-12 show G-, O⁶-MeG- and O⁶-CMG-containing duplex ODNs, respectively; lanes 1, 5 and 9 show untreated ODN (7 pmoles); lanes 2, 6 and 10 show ODN incubated with MGMT (21 pmoles) only, and then digested with BsaJI or PstI; lanes 3, 7 and 11 show ODN incubated with BsaJI or PstI only; and lane 4, 8 and 12 show ODN incubated with MGMT and then digested with BsaJI or PstI. Non-denaturing PAGE gels were imaged as described in Materials and Methods.

Figure 4.

MALDI-TOF mass spectral analysis of tryptic (Tp) peptides of unmodified MGMT (A; control) or MGMT incubated with ODNs containing O⁶-MeG (B) or O⁶-CMG (C). Modified peptides (Tp20* MeG and Tp20** CMG) are observed at m/z 1329.7 and 1372.7 in (B) and (C), respectively. MBP describes tryptic peptides derived from the MBP affinity tag on MGMT.