DNA Polymerase λ Active Site Favors a Mutagenic Mispair between the Enol Form of Deoxyguanosine Triphosphate Substrate and the Keto Form of Thymidine Template: A Free Energy Perturbation Study

Human
DNA polymerase λ is an intermediate fidelity member
of the X family, which plays a role in DNA repair. Recent X-ray diffraction
structures of a ternary complex of a loop-deletion mutant of polymerase
λ, a deoxyguanosine triphosphate analogue, and a gapped DNA
show that guanine and thymine form a mutagenic mispair with an unexpected
Watson–Crick-like geometry rather than a wobble geometry. Hence,
there is an intriguing possibility that either thymine in the DNA
or guanine in the deoxyguanosine triphosphate analogue may spend a
substantial fraction of time in a deprotonated or enol form (both
are minor species in aqueous solution) in the active site of the polymerase
λ mutant. The experiments do not determine particular forms
of the nucleobases that contribute to this mutagenic mispair. Thus,
we investigate the thermodynamics of formation of various mispairs
between guanine and thymine in the ternary complex at a neutral pH
using classical molecular dynamics simulations and the free energy
perturbation method. Our free energy calculations, as well as a comparison
of the experimental and computed structures of mispairs, indicate
that the Watson–Crick-like mispair between the enol tautomer
of guanine and the keto tautomer of thymine is dominant. The wobble
mispair between the keto forms of guanine and thymine and the Watson–Crick-like
mispair between the keto tautomer of guanine and the enol tautomer
of thymine are less prevalent, and mispairs that involve deprotonated
guanine or thymine are thermodynamically unlikely. These findings
are consistent with the experiment and relevant for understanding
mechanisms of spontaneous mutagenesis.