An 8-oxo-guanine repair pathway coordinated by MUTYH glycosylase and DNA polymerase λ
Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved August 31, 2009
Abstract
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.
Acknowledgments.
We thank G.L. Dianov, J.L. Parsons, I. Shevelev, and A.L. Lu for providing expression constructs; E. Ferrari and R. Imhof for purifying DNA pol λ, FEN1, and DNA ligase III; and G. Villani, G. Maga, K. Ramadan, and M. Stucki for critically reading of the manuscript and their suggestions. This work is supported by the Swiss National Science Foundation Grant 3100–109312/2 and the University of Zürich.
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Received: June 30, 2009
Published online: October 27, 2009
Published in issue: October 27, 2009
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Acknowledgments
We thank G.L. Dianov, J.L. Parsons, I. Shevelev, and A.L. Lu for providing expression constructs; E. Ferrari and R. Imhof for purifying DNA pol λ, FEN1, and DNA ligase III; and G. Villani, G. Maga, K. Ramadan, and M. Stucki for critically reading of the manuscript and their suggestions. This work is supported by the Swiss National Science Foundation Grant 3100–109312/2 and the University of Zürich.
Notes
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0907280106/DCSupplemental.
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The authors declare no conflict of interest.
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An 8-oxo-guanine repair pathway coordinated by MUTYH glycosylase and DNA polymerase λ, Proc. Natl. Acad. Sci. U.S.A.
106 (43) 18201-18206,
https://doi.org/10.1073/pnas.0907280106
(2009).
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