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8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins

Nature volume 447, pages 606–608 (2007)Cite this article

Abstract

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells1. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols—belonging to the B, Y and X classes—during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential2,3,4. A major and specific effect was found for 8-oxo-G bypass with DNA pols λ and η. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol λ, and 68-fold by DNA pol η, respectively. Experiments with DNA-pol-λ-null cell extracts suggested an important role for DNA pol λ. On the other hand, DNA pol ι, together with DNA pols α, δ and β, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.

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Figure 1: PCNA and RP-A influence bypass of 8-oxo-G by human DNA pol λ.
Figure 2: 8-oxo-G bypass by human DNA pols η and ι with RP-A and PCNA.

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Acknowledgements

We thank R. Woodgate for his generous gift of recombinant human DNA pol η and ι. U.H. and U.W. are supported by the Swiss National Science Foundation, by the UBS “im Auftrag eines Kunden”, and U.H. and E.F. by the University of Zürich, which gave a grant in aid to G.M. G.M. is supported partially by the CARIPLO Foundation Project “Oncogenetica e Proteomica della Replicazione”. G.V. is supported by CNRS and ARC.

Author Contributions G.M. and U.H. had the original idea. G.M. supervised the overall experimental strategy and performed all the experiments with human DNA pol λ and cell extracts; E.C. performed all the experiments with human DNA polymerases α, δ, β, ι and η; U.W. and B.B. generated the DNA POLL+/+ and POLL-/- MEFs and characterized their phenotype; G.V. provided the damaged templates; E.F. purified DNA pols α, δ, PCNA and RP-A; G.M., G.V. and U.H. designed and interpreted all the experiments and equally contributed to manuscript writing and figures preparation.

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Authors and Affiliations

  1. Institute of Molecular Genetics IGM-CNR, via Abbiategrasso 207, I-27100 Pavia, Italy,

    Giovanni Maga & Emmanuele Crespan

  2. Institute de Pharmacologie et de Biologie Structurale, IPBS-CNRS, Centre National de la Recherche Scientifique, Route de Narbonne 205, 31077 Toulouse, France,

    Giuseppe Villani

  3. Institute National Français de Recherche Médicale, Université Paris Descartes, Faculté de Médecine René Descartes, Site Necker-Enfants Malades, 156 rue de Vaugirard 75730 Paris, Cedex 15, France,

    Barbara Bertocci

  4. Institute for Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland,

    Giovanni Maga, Ursula Wimmer, Elena Ferrari & Ulrich Hübscher

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  1. Giovanni Maga
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Correspondence to Giovanni Maga.

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Supplementary information

Supplementary Information

This file contains Supplementary Figures S1-S5 with Legends and Supplementary Table S1. The Supplementary Figures provide additional evidences for the specificity of the observed effects of PCNA and RP-A on DNA polymerases lambda and eta during 8-oxo-G bypass, including results with other lesions such as abasic site and cis-platinum adduct.The Supplementary Table 1 summarizes all the kinetic constants for nucleotide incorporation by DNA polymerases lambda, alpha eta and iota opposite a normal G or an 8-oxo-G. (PDF 977 kb)

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Maga, G., Villani, G., Crespan, E. et al. 8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins. Nature 447, 606–608 (2007). https://doi.org/10.1038/nature05843

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