Mismatch repair systems might facilitate the chromosomal recombination induced by N-nitrosodimethylamine, but not by N-nitrosodiethylamine, in Drosophila.

Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(-/-)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/-)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(-/-) flies than in spel1(+/-) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(-/-) flies than in spel1(+/-) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.

Distinct pathways for repairing mutagenic lesions induced by methylating and ethylating agents.

DNA alkylation damage can be repaired by nucleotide excision repair (NER), base excision repair (BER) or by direct removal of alkyl groups from modified bases by O(6)-alkylguanine DNA alkyltransferase (AGT; E.C. 2.1.1.63). DNA mismatch repair (MMR) is also likely involved in this repair. We have investigated alkylation-induced mutagenesis in a series of NER- or AGT-deficient Escherichia coli strains, alone or in combination with defects in the MutS, MutL or MutH components of MMR. All strains used contained the F'prolac from strain CC102 (F'CC102) episome capable of detecting specifically lac GC to AT reverse mutations resulting from O(6)-alkylguanine. The results showed the repair of O(6)-methylguanine to be performed by AGT ≫ MMR > NER in order of importance, whereas the repair of O(6)-ethylguanine followed the order NER > AGT > MMR. Studies with double mutants showed that in the absence of AGT or NER repair pathways, the lack of MutS protein generally increased mutant frequencies for both methylating and ethylating agents, suggesting a repair or mutation avoidance role for this protein. However, lack of MutL or MutH protein did not increase alkylation-induced mutagenesis under these conditions and, in fact, reduced mutagenesis by the N-alkyl-N-nitrosoureas MNU and ENU. The combined results suggest that little or no alkylation damage is actually corrected by the mutHLS MMR system; instead, an as yet unspecified interaction of MutS protein with alkylated DNA may promote the involvement of a repair system other than MMR to avoid a mutagenic outcome. Furthermore, both mutagenic and antimutagenic effects of MMR were detected, revealing a dual function of the MMR system in alkylation-exposed cells.

Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency.

The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O(6)-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O(6)-MeG:T, O(6)-MeG:C, O(6)-EtG:T, O(6)-EtG:C, G:T and G:C. Dissociation constants (K(d)), which reflect the strength of binding, followed the order G:T->O(6)-MeG:T->O(6)-EtG:T-=O(6)-EtG:C-> or =O(6)-MeG:C->G:C. These results suggest that a thymine base paired with O(6)-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O(6)-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.

Novel antimutagenic factors derived from the edible mushroom Agrocybe cylindracea.

Studies have shown that certain foods contain compounds with antigenotoxic activities. Here, we ask if dried powders and/or extracts from three edible mushrooms, Agrocybe cylindracea, Lentinula edodes and Pleurotus ostreatus, have a mitigating effect on genotoxicity. We used two in vivo assays: the Drosophila DNA repair test and the Drosophila wing spot test (also known as SMART) which measures somatic mutation and recombination. Eight carcinogens were tested with the mushroom powders: 2-AAF, aflatoxin B1, DMBA, IQ, MeIQx, MNU NDMA, and 4NQO. We found that A. cylindracea and P. ostreatus powders can suppress DNA damage induced by each of the mutagens we tested. In contrast, L. edodes has an inhibitory effect on DNA damage induced by only a sub-set of mutagens, namely aflatoxin B1, NDMA, MNU and 4NQO. In addition, A. cylindracea extracts were able to suppress somatic cell mutation induced by aflatoxin B1, MMC, MNU, NDMA, NMOR and 4NQO. These results suggest that Agrocybe genus mushrooms contain factors with antigenotoxic activity, including anti-recombinogenic activity. Furthermore, the antigenotoxic activity of A. cylindracea powder can be extracted in water but not in ethyl acetate or methanol, and is sensitive to heat treatment. The data suggest that there is a novel antigenotoxic factor(s) in A. cylindracea, possibly in the form of a peptide or protein.