Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 103, Issue 52, Pages 19701-19706Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0609580103
Keywords
crystal structure; DNA damage; DNA polymerase; protein-DNA complex
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Funding
- NCI NIH HHS [P01 CA092584, 5 P01 CA92584] Funding Source: Medline
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Methylating agents are widespread environmental carcinogens that generate a broad spectrum of DNA damage. Methylation at the guanine 06 position confers the greatest mutagenic and carcinogenic potential. DNA polymerases insert cytosine and thymine with similar efficiency opposite O-6-methyl-guanine (O6MeG). We combined pre-steady-state kinetic analysis and a series of nine x-ray crystal structures to contrast the reaction pathways of accurate and mutagenic replication of O6MeG in a high-fidelity DNA polymerase from Bacillus stearothermophilus. Polymerases achieve substrate specificity by selecting for nucleoticles with shape and hydrogen-bonding patterns that complement a canonical DNA template. Our structures reveal that both thymine and cytosine O6MeG base pairs evade proofreading by mimicking the essential molecular features of canonical substrates. The steric mimicry depends on stabilization of a rare cytosine tautomer in C center dot O6MeG-polymerase complexes. An unusual electrostatic interaction between O-methyl protons and a thymine carbonyl oxygen helps stabilize T-O6MeG pairs bound to DNA polymerase. Because DNA methylators constitute an important class of chemotherapeutic agents, the molecular mechanisms of replication of these DNA lesions are important for our understanding of both the genesis and treatment of cancer.
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