Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 5, Pages 1862-1867Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1012825108
Keywords
mutagenesis; replication fidelity; nascent base pair; mispair
Categories
Funding
- US Department of Energy, Office of Science, Office of Basic Energy Sciences [W-31-109-Eng-38]
- Division of Intramural Research of the National Institutes of Health, National Institute of Environmental Health Sciences [Z01 ES065070]
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In describing the DNA double helix, Watson and Crick suggested that spontaneous mutation may be due to a base occasionally occurring in one of its less likely tautomeric forms. Indeed, among many mispairing possibilities, either tautomerization or ionization of bases might allow a DNA polymerase to insert a mismatch with correct Watson-Crick geometry. However, despite substantial progress in understanding the structural basis of error prevention during polymerization, no DNA polymerase has yet been shown to form a natural base-base mismatch with Watson-Crick-like geometry. Here we provide such evidence, in the form of a crystal structure of a human DNA polymerase lambda variant poised to misinsert dGTP opposite a template T. All atoms needed for catalysis are present at the active site and in positions that overlay with those for a correct base pair. The mismatch has Watson-Crick geometry consistent with a tautomeric or ionized base pair, with the pH dependence of misinsertion consistent with the latter. The results support the original idea that a base substitution can originate from a mismatch having Watson-Crick geometry, and they suggest a common catalytic mechanism for inserting a correct and an incorrect nucleotide. A second structure indicates that after misinsertion, the now primer-terminal G.T mismatch is also poised for catalysis but in the wobble conformation seen in other studies, indicating the dynamic nature of the pathway required to create a mismatch in fully duplex DNA.
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