O6-methylguanine induces altered proteins at the level of transcription in human cells.
O(6)-Methylguanine (O(6)-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 O(6)-meG on transcription in human cells and investigated the subsequent effects on protein function following translation of the resulting mRNA. In HEK293 cells, O(6)-meG induced incorporation of uridine or cytidine in nascent RNA opposite the adduct. In cells containing active O(6)-alkylguanine-DNA alkyltransferase (AGT), which repairs O(6)-meG, 3% misincorporation of uridine was observed opposite the lesion. In cells where AGT function was compromised by addition of the AGT inhibitor O(6)-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 O(6)-meG is highly mutagenic at the level of transcription in human cells, leading to an altered protein load, especially when AGT is inhibited.
Burns
John A
JA
Department of Biology, New York University, 1009 Silver Center, 100 Washington Square East, New York, NY 10003, USA.
Dreij
Kristian
K
Cartularo
Laura
L
Scicchitano
David A
DA
D004261:DNA Replication; D005456:Fluorescent Dyes / Q000032:analysis; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D016296:Mutagenesis; D010957:Plasmids / Q000096:biosynthesis / Q000737:chemistry; D014158:Transcription, Genetic; D000097573:Red Fluorescent Protein
D005456:Fluorescent Dyes; D006147:Guanine; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C008449:O-(6)-methylguanine