8-oxo-guanine repair pathway coordinated by MUTYH glycosylase and DNA polymerase λ

• Published in: Proceedings of the National Academy of Sciences - PNAS 2009-10, Vol.106 (43), p.18201-18206
• Main Authors: van Loon, Barbara, Hübscher, Ulrich
• Format: Article
• Language: English
• Subjects: Biological Sciences; Cell extracts; Cell growth; Cell nucleus; DNA; DNA Damage; DNA Glycosylases - genetics; DNA Glycosylases - metabolism; DNA Ligase ATP; DNA Ligases - metabolism; DNA Polymerase beta - genetics; DNA Polymerase beta - metabolism; DNA Repair; DNA Replication; Genomic Instability; Guanine - analogs & derivatives; Guanine - metabolism; HeLa Cells; Humans; Lesions; Mutagenesis; Nuclear antigens; Oxidation-Reduction; Proliferating Cell Nuclear Antigen - metabolism; Protein Binding; Reactive Oxygen Species - metabolism; Stem cells; Substrate Specificity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0907280106

Reactive oxygen species (ROS) interact with DNA, frequently generating highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxo-G) lesions. Replicative DNA polymerases (pols) often misincorporate adenine opposite 8-oxo-G. The subsequent repair mechanism allowing the removal of adenine and formation of C:8-oxo-G base pair is essential to prevent C:G to A:T transversion mutations. Here, we show by immunofluorescence experiments, in cells exposed to ROS, the involvement of MutY glycosylase homologue (MUTYH) and DNA pol λ in the repair of A:8-oxo-G mispairs. We observe specific recruitment of MUTYH, DNA pol λ, proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN1) and DNA ligases I and III from human cell extracts to A:8-oxo-G DNA, but not to undamaged DNA. Using purified human proteins and a DNA template, we reconstitute the full pathway for the faithful repair of A:8-oxo-G mispairs involving MUTYH, DNA pol λ, FEN1, and DNA ligase I. These results reveal a cellular response pathway to ROS, important to sustain genomic stability and modulate carcinogenesis.