The in vivo mutagenic frequency and specificity of O6-methylguanine in phi X174 replicative form DNA.

Abstract

A bacteriophage 4X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented. A (-)-strand primer containing 06 methylguanine in a specific site was hybridized to a singlestranded region in gene G of OX gapped duplex DNA. The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells. All gene G mutants generated by the modification were rescued by genetic complementation. An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand. In each mutantcontaining infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type. Thus, in vivo, 06-methylguanine in replicating OX DNA has a mutagenic frequency of 75%. When repair of 06 methylguanine occurred, it was prereplicative. The mutations were due exclusively to the misincorporation of thymine.Most chemical carcinogens are mutagens (1) that react with DNA to form various covalent adducts (2, 3). To establish a cause-and-effect relationship between the specific DNA lesions and the specific mutations produced, we developed a system for assaying site-specific mutagenesis in gene G of bacteriophage 4X174 (4, 5). Gene G codes for the phage spike protein, which is essential for the formation of infectious phage. The system produced mutant phage at low percentage yield (4) due to large numbers of(+)-strand-derived wild-type (wt) phage. This also distorts the true percentage of any wt phage produced from the adduct-containing (-) strand. Low mutant recovery was a general problem in the study of site-specific mutagenesis, and strategies have been developed in different systems to increase mutant yields (6-10).We here describe a version of the 4X174-based mutagenesis system. A complementation plasmid efficiently supports growth of gene G mutants, and an amber (am) mutation in the lysis gene E of the (+) strand of the transforming OX

Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O 6 -alkylguanine adducts in Escherichia coli

“…Briefly, upon introduction into E. coli cells, the O 6 -alkylguanine adduct located within the unique SmaI restriction site contained in the plasmid is processed by repair pathways and/or undergoes replication. Due to the high miscoding capacity of these adducts (1)(2)(3)(4), unrepaired adducts will yield plasmid progeny containing a majority of G→A transitions at the adduct site, thus inactivating the SmaI restriction site. With single-stranded constructs, the mutant fraction may reach 100% if the adduct is fully miscoding.…”

“…Despite their low abundance, O 6 -alkylguanine adducts are responsible for most biological consequences induced by alkylating agents, both in terms of mutagenesis and toxicity. Whereas mutagenesis results from the capacity of these adducts to mispair efficiently with T during replication (1)(2)(3)(4), the way these adducts induce toxicity is more cumbersome. Indeed, cytotoxicity involves the mismatch repair (MMR) system that recognizes the mutagenic O 6 -methylguanine: T (mG:T) replication intermediates.…”

Mismatch repair proteins collaborate with methyltransferases in the repair of O6-methylguanine

“…The net biological impact of a lesion depends on the balance between the ability of the cell to repair the lesion and the mutagenic and toxic properties of the lesion. One particularly critical type of DNA alkylation damage is O 6 -methylguanine (O 6 mG), which has been shown to be both mutagenic [1][2][3][4] and, when cells cannot distinguish nascent from parental DNA strands, toxic [5][6][7]. Extensive in vivo mutagenesis studies done in our laboratory and others established that O 6 mG codes as adenine during replication in E. coli nearly 100% of the time [1,8].…”

Use of high throughput sequencing to observe genome dynamics at a single cell level

“…On average, 70 mutations per genome were observed. The overwhelming majority of the changes are G:C 3 A:T transitions, well in accord with the known mutagenic specificity of EMS (15)(16)(17)(18)(19). The genome-wide distribution of the newly acquired mutations induced by EMS in the CC102 strain This article is a PNAS Direct Submission.…”

“…Fig. 3A (Top) shows the standard model of the EMS-induced O 6 alkyl guanine specifically mispairing with thymine, resulting in a G to A replacement upon the second round of replication (15)(16)(17)(18)(19). Considering now a continuous stretch of DNA, and assuming that each strand in the original DNA is randomly affected by EMS, one would expect that the segregation between daughter strands into different cells after replication will lead to each descendant cell having exclusively G 3 A or C 3 T conversions (Fig.…”