Abstract
Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.
Highlights
Oxidized dGTP (8-oxo-7,8-dihydro-2 ́-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease
Structural characterization of 8-oxodGTP insertion has only been reported for X-family pols β5,11–14 and λ15, and insertion opposite Ct is limited to pol β12
(Supplementary Fig. 1 and Supplementary Table 1), 8oxodGTP was preferentially inserted opposite At (~25-fold) in the presence of Mg2+. This specificity arose from an increase in efficiency of 8-oxodGTP insertion opposite At, and decreased efficiency opposite Ct, compared to dGTP insertion
Summary
Oxidized dGTP (8-oxo-7,8-dihydro-2 ́-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anticonformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair. The results uncover unique molecular strategies for modulation of oxidized and undamaged dGTP insertion by this X-family DSB repair polymerase
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