Capturing a mammalian DNA polymerase extending from an oxidized nucleotide

Abstract

The oxidized nucleotide, 8-oxo-7,8-dihydro-2΄-deoxyguanosine (8-oxoG), is one of the most abundant DNA lesions. 8-oxoG plays a major role in tumorigenesis and human disease. Biological consequences of 8-oxoG are mediated in part by its insertion into the genome, making it essential to understand how DNA polymerases handle 8-oxoG. Insertion of 8-oxoG is mutagenic when opposite adenine but not when opposite cytosine. However, either result leads to DNA damage at the primer terminus (3΄-end) during the succeeding insertion event. Extension from DNA damage at primer termini remains poorly understood. Using kinetics and time-lapse crystallography, we evaluated how a model DNA polymerase, human polymerase β, accommodates 8-oxoG at the primer terminus opposite cytosine and adenine. Notably, extension from the mutagenic base pair is favored over the non-mutagenic base pair. When 8-oxoG is at the primer terminus opposite cytosine, DNA centric changes lead to a clash between O8 of 8-oxoG and the phosphate backbone. Changes in the extension reaction resulting from the altered active site provide evidence for a stabilizing interaction between Arg254 and Asp256 that serves an important role during DNA synthesis reactions. These results provide novel insights into the impact of damage at the primer terminus on genomic stability and DNA synthesis.

Visualizing Rev1 Catalyze Protein-template DNA Synthesis

“…To observe the Rev1 catalytic cycle at the atomic level, we employed time-lapse X-ray crystallography ( Fig. 1A) (24)(25)(26)(27)(28)(29). To accomplish this, we first generate binary Rev1/DNA complex crystals with a templating guanine.…”

“…These binary Rev1/DNA crystals are subsequently transferred into a cryoprotectant solution containing dCTP and CaCl2 to generate Rev1 ground-state ternary (Rev1/DNA/dCTP) complexes. Importantly, Ca 2+ facilitates nucleotide binding but does not support catalysis (24)(25)(26)(27)(28)(29). To initiate catalysis, the Rev1 ground-state ternary complex crystals are subsequently transferred to a cryoprotectant solution containing either MgCl2 or MnCl2.…”

“…Interestingly, the third metal seen in the product state of other polymerases studied by time-lapse crystallography has been proposed to play a role in facilitating catalysis, stabilizing the product state, and preventing the reverse reaction (24)(25)(26)(27)(28)(29). Therefore, the breakdown of pyrophosphate by Rev1 could explain why a third metal is not seen during Rev1-mediated insertion.…”

Reading and Misreading 8-oxoguanine, a Paradigmatic Ambiguous Nucleobase

“…Pol β and Pol λ are among few polymerases for which incorporation of oxodGMP has been structurally characterized [262][263][264][265]. These structures illuminate general principles of non-mutagenic and mutagenic oxodGMP insertion and extension.…”

“…In contrast to the incorporation opposite oxoG, X-family Pol β and Pol λ have a clear preference both for forming pro-mutagenic A:oxoG pairs with the oxodGTP substrate [197,198,[262][263][264] and for extension from such primer termini [265]. Pol β and Pol λ incorporate oxodGMP opposite A with 11-40-fold preference over C [197,198,[262][263][264].…”