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Dpo4 is hindered in extending a G·T mismatch by a reverse wobble

Nature Structural & Molecular Biology volume 11, pages 457–462 (2004)Cite this article

Abstract

The ability or inability of a DNA polymerase to extend a mispair directly affects the establishment of genomic mutations. We report here kinetic analyses of the ability of Dpo4, a Y-family polymerase from Sulfolobus solfataricus, to extend from all mispairs opposite a template G or T. Dpo4 is equally inefficient at extending these mispairs, which include, surprisingly, a G·T mispair expected to conform closely to Watson-Crick geometry. To elucidate the basis of this, we solved the structure of Dpo4 bound to G·T-mispaired primer template in the presence of an incoming nucleotide. As a control, we also determined the structure of Dpo4 bound to a matched A-T base pair at the primer terminus. The structures offer a basis for the low efficiency of Dpo4 in extending a G·T mispair: a reverse wobble that deflects the primer 3′-OH away from the incoming nucleotide.

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Figure 1: Dpo4-catalyzed extension of a G·T primer–terminal mispair.
Figure 2: Dpo4 in complex with matched and mismatched primer termini.
Figure 3

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Acknowledgements

We thank the staff at the Advanced Photon Source and the National Synchrotron Light Source for facilitating X-ray data collection. We thank T. Edwards for help with data collection and processing. This was supported was supported by US National Institutes of Health grant CA094006 (A.K.A. and L.P.).

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Authors and Affiliations

  1. Department of Physiology and Biophysics, Structural Biology Program, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, 10029, New York, USA

    Jose Trincao, Carlos R Escalante & Aneel K Aggarwal

  2. Sealy Center for Molecular Science, University of Texas Medical Branch, 6.014 Medical Research Building, 11 and Mechanic Streets, Galveston, 77755, Texas, USA

    Robert E Johnson, William T Wolfle, Satya Prakash & Louise Prakash

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  1. Jose Trincao
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Correspondence to Aneel K Aggarwal.

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Trincao, J., Johnson, R., Wolfle, W. et al. Dpo4 is hindered in extending a G·T mismatch by a reverse wobble. Nat Struct Mol Biol 11, 457–462 (2004). https://doi.org/10.1038/nsmb755

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