Abstract
DNA-damage-induced SOS mutations arise when Escherichia coli DNA polymerase (pol) V, activated by a RecA nucleoprotein filament (RecA*), catalyses translesion DNA synthesis. Here we address two longstanding enigmatic aspects of SOS mutagenesis, the molecular composition of mutagenically active pol V and the role of RecA*. We show that RecA* transfers a single RecA–ATP stoichiometrically from its DNA 3′-end to free pol V (UmuD′2C) to form an active mutasome (pol V Mut) with the composition UmuD′2C–RecA–ATP. Pol V Mut catalyses TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecA–ATP in the complex. Reactivation of pol V Mut is triggered by replacement of RecA–ATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecA–ATP to pol V, and thus generate active mutasomal complex for translesion synthesis.
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Acknowledgements
This work was supported by National Institutes of Health grants to M.F.G. (ES12259; R37GM21422) and M.M.C. (GM32335), and funds from the NICHD/NIH Intramural Research Program to K.K. and R.W. The authors thank R. Britt for preparation of the RecA E38KΔC17 protein used in this study. MALS data were collected using the USC NanoBiophysics Core Facility, with the aid and cooperation of N. Chelyapov. We thank J. Bertram for his help in performing the MALS experiment and analysing the data. We thank J. Petruska for his comments.
Author Contributions Q.J. performed the experiments and experimental analyses; K.K. performed the construction and purification of His-tagged pol V; M.M.C. provided the purified RecA E38KΔC17 protein; Q.J. and M.F.G. designed the experiments with input from M.M.C. and R.W.; M.F.G., Q.J., M.M.C. and R.W. wrote the manuscript.
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This file contains Supplementary Methods and Supplementary Figures 1-9. Supplementary Figures were corrected on 23 July, 2009. (PDF 1379 kb)
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Jiang, Q., Karata, K., Woodgate, R. et al. The active form of DNA polymerase V is UmuD′2C–RecA–ATP. Nature 460, 359–363 (2009). https://doi.org/10.1038/nature08178
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DOI: https://doi.org/10.1038/nature08178
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