Methylating agents are widespread environmental carcinogens that generate a
broad spectrum of DNA damage. Methylation at the guanine O(6) 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
nucleotides 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.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.
Figure 1.
Fig. 1. Structures of O6MeG·C (a) and O6MeG·T
(b) pairs in DNA duplexes, unbound by protein. Hydrogen bonds
are shown as dashed lines. (a Left) O6MeG·C wobble pair
from ref. 27. (a Right) Model of Watson–Crick
O6MeG·protonated C pair from ref. 52. (b) O6MeG·T
pairs from refs. 29 and 30. The presence or absence of the H
bond indicated by the "?" has been the subject of some
controversy (29, 30).
Figure 3.
Fig. 3. T·O6MeG (a) and C·O6MeG (b)
conformations in the –2 base pair position. Mesh shows 1 2 F[o]
– F[c] electron density for the base pairs.
2 F[o]
– F[c] electron density for the base pairs.
