Article
- The EMBO Journal (2005) 24, 2957 - 2967
- doi:10.1038/sj.emboj.7600786
Published online: 18 August 2005
Subject Categories:
Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis
Alexandra Vaisman1, Hong Ling2,a, Roger Woodgate1 and Wei Yang2
- Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
Correspondence to:
Wei Yang, Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Tel.: +1 301 402 4645; Fax: +1 301 496 0201; E-mail: Wei.Yang@nih.gov
aPresent address: Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1
Received 4 May 2005; Accepted 22 July 2005
Abstract
We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not.
Keywords:
- base stacking,
- fidelity,
- metal ion,
- mismatch,
- pyrophosphorolysis
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