ja5b02109_si_001.pdf (1.41 MB)
Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography
journal contribution
posted on 2015-04-22, 00:00 authored by Rajan Vyas, Andrew
J. Reed, E. John Tokarsky, Zucai SuoOne common oxidative DNA lesion,
8-oxo-7,8-dihydro-2′-deoxyguanine
(8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson–Crick base pair with correct
deoxycytidine 5′-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine
5′-triphosphate (dATP). Here, we utilized time-resolved X-ray
crystallography to follow 8-oxoG bypass by human DNA polymerase β
(hPolβ). In the 12 solved structures, both Watson–Crick
(anti-8-oxoG:anti-dCTP) and Hoogsteen
(syn-8-oxoG:anti-dATP) base pairing
were clearly visible and were maintained throughout the chemical reaction.
Additionally, a third Mg2+ appeared during the process
of phosphodiester bond formation and was located between the reacting
α- and β-phosphates of the dNTP, suggesting its role in
stabilizing reaction intermediates. After phosphodiester bond formation,
hPolβ reopened its conformation, pyrophosphate was released,
and the newly incorporated primer 3′-terminal nucleotide stacked,
rather than base paired, with 8-oxoG. These structures provide the
first real-time pictures, to our knowledge, of how a polymerase correctly
and incorrectly bypasses a DNA lesion.