O 6-Methylguanine induces altered proteins at the level of transcription in human cells

Abstract

O6-Methylguanine (O6-meG), which is produced in DNA following exposure to methylating agents, instructs human RNA polymerase II to mis-insert bases opposite the lesion during transcription. In this study, we examined the effect of O6-meG on transcription in human cells and investigated the subsequent effects on protein function following translation of the resulting mRNA. In HEK293 cells, O6-meG induced incorporation of uridine or cytidine in nascent RNA opposite the adduct. In cells containing active O6-alkylguanine-DNA alkyltransferase (AGT), which repairs O6-meG, 3% misincorporation of uridine was observed opposite the lesion. In cells where AGT function was compromised by addition of the AGT inhibitor O6-benzylguanine, ∼58% of the transcripts contained a uridine misincorporation opposite the lesion. Furthermore, the altered mRNA induced changes to protein function as demonstrated through recovery of functional red fluorescent protein (RFP) from DNA coding for a non-fluorescent variant of RFP. These data show that O6-meG is highly mutagenic at the level of transcription in human cells, leading to an altered protein load, especially when AGT is inhibited.

Effects of 6-Thioguanine and S6-Methylthioguanine on Transcription in Vitro and in Human Cells

“…These findings indicate that, when located on the template strand of an actively transcribed gene, S 6 mG is primarily repaired by TC-NER in human cells. Similar to our results, it was reported that O 6 -methylguanine, a structural analog of S 6 mG, contributes to mutagenesis and blockage of transcription and thus might invoke TC-NER (40,41). Previous replication studies have also shown that S 6 mG is capable of inducing high frequencies of G 3 A transition in E. coli and mammalian cells (21,22).…”

“…Previous studies have provided some clues that the mutations induced by S 6 mG and S G during replication might have a role in the carcinogenicity of thiopurine drugs (21,22). In this vein, transcriptional mutagenesis has also been proposed as one of the principal inducers of cancer and other human diseases, despite the lack of direct evidence linking transcriptional mutations to cancer (33,40,52,53). Notably, a previous study has provided important implications for the role of transcriptional mutagenesis in tumorigenesis in that mutagenic transcriptional bypass of 8-oxoguanine could lead to activation of an oncogenic pathway (53).…”

The Functions of Serine 687 Phosphorylation of Human DNA Polymerase η in UV Damage Tolerance

“…At 24 h after transfection, the progenies of the plasmid were isolated by using a modified alkali lysis method (30). The residual unreplicated plasmid was further processed by combined digestion with DpnI and exonuclease III, as described previously (31)(32)(33). The progeny genomes arising from in vivo replication were PCR amplified with primers spanning the lesion site; the resulting PCR products were restriction-digested with NcoI and SfaNI and subsequently analyzed by PAGE and LC-MS/MS, as described elsewhere (28,29).…”

Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis

“…For direct reversal of O 6 -MeG, a plasmid that encodes a nonfluorescent protein in the absence of the lesion was prepared. Introduction of a site-specific O 6 -MeG lesion into the transcribed strand causes transcription errors (31) DRC for three pathways, namely NER, MMR, and the direct reversal of O 6 -MeG (MGMT) was measured in five lymphoblastoid cell lines (Fig. 2).…”