Certain hexavalent chromium [Cr(VI)] compounds are human lung carcinogens. Although much is known about Cr-induced DNA damage, very little is known about mechanisms of Cr(VI) mutagenesis and the role that DNA repair plays in this process. Therefore, the goal of this research was to investigate the role of excision repair pathways in Cr(VI)-mediated mutagenesis using S. cerevisiae and mammalian cells deficient in various components of nucleotide excision repair (NER), base excision repair (BER) and translesion synthesis (TLS). Repair-proficient Chinese hamster ovary (CHO) cells (AA8), NER-deficient (UV-5 and UV-41) and BER-inhibited cells were treated with Cr(VI) and monitored for forward mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locus. BER was inhibited using methoxyamine hydrochloride (Mx), which binds to apurinic/abasic (AP) sites generated during BER. Notably, we found that both NER-deficient (UV-5, UV-41) and BER-inhibited (AA8 + Mx) cells displayed attenuated Cr(VI) mutagenesis. To determine whether this was unique to Cr(VI), we included the alkylating agent, methylmethane sulfonate (MMS) and ultraviolet radiation (260 nm) in our studies. Similar to Cr(VI), UV-5 cells exhibited a marked attenuation of MMS mutagenesis, but were hypermutagenic following UV exposure. Because there are a limited number of excision repair (ER)-deficient CHO cells, S. cerevisiae was employed to verify whether the attenuated mutagenesis was limited to UV-5 and UV-41 deficient and BER-inhibited CHO cells or if the attenuation was the result of an underlying mechanism associated with NER and BER. Three different parental strains of S. cerevisiae (SJR751, SJR922 and BY4741) were used to assay different components of NER and BER and to help elucidate a mechanism of ER-dependent chemical mutagenesis. NER-deficient yeast (rad1, rad2 and rad14) exhibited attenuated forward mutagenesis at the CAN1 locus following treatment with Cr(VI) and MMS. As with the UV exposed NER-deficient CHO cells, rad1 yeast were also hypermutagenic to UV radiation (260 nm). Various BER-deficient strains (apn1, ntg1, ntg2, ntg1/2 ) strains additionally exhibited attenuated mutagenesis, genetically supporting the pharmacologically inhibited BER (Mx) attenuated mutagenesis found in mammalian cells. Taken together, there appears to be an underlying mechanism involving ER and chemical mutagenesis. TLS is involved in the bypass of damaged DNA during DNA replication, potentially at the cost of mutations. Using TLS-(rev3 and rad30) and NER/TLS- (rad1, rad1 rev3 and rad1 rad30) deficient yeast, we made the novel observation that NER (rad1) is functioning in coordination with TLS error-prone bypass polymerase (rev3). Moreover, both NER (rad1) and TLS (rev3) are required for chemical mutagenesis. Interestingly, homologous recombination (HR)-deficient (rad52) mutants exhibited a wild-type level of mutagenesis following treatment with either Cr(VI) or MMS. Due to the association observed in the NER/TLS mutants, it is suggested that NER and TLS are still functional in HR-deficient yeast. Although it has been historically thought that the loss of DNA repair will increase chemical mutagenesis, this study has provided novel insight to a mechanism by which DNA repair is responsible for chemical mutagenesis. The results presented in this study have broad regulatory and therapeutic implications affecting the way we currently think about environmental and occupational chemical exposures and chemotherapy. NER is a repair mechanism observed in all living organisms, as such, these data suggest that NER may potentially serve as a pro-mutagenic survival pathway involved in evolution.Using TLS-(rev3 and rad30) and NER/TLS- (rad1, rad1 rev3 and rad1 rad30) deficient yeast, we made the novel observation that NER (rad1) is functioning in coordination with TLS error-prone bypass polymerase (rev3).
|Title||:||Going Against the Grain: DNA Excision Repair is Required for Genotoxic Mutagenesis|
|Author||:||Bradford Raymond Brooks|
|Publisher||:||ProQuest - 2008|