Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide

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.

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

“…The major differences between conformations was provided by Lys325 for the unreactive orientation (−34.5 kcal mol −1 ) compared to the reacted active site (−5.2 kcal mol −1 ). Sugar stabilization was provided by Trp434 in the reacted active site (−4.4 kcal mol −1 compared to 0.8 kcal mol −1 ), while Lys438 stabilized the ribose sugar in the unreactive orientation (−17.3 kcal mol −1 compared to −6.7 kcal mol −1 ), consistent with that observed in the Mn 2+ -ground state 8-oxo-dGTP(anti):C t ternary complex 24 . While Gln441 does not directly interact with either incoming oxidized deoxy- or ribonucleotide (Fig.…”

“…2b , c). As we previously showed, 8-oxo-dGTP forms hydrogen bonds with C t in the anti-conformation, where O3´ is poised to attack P α in the precatalytic ternary complex 24 . While the triphosphate of the incoming 8-oxo-rGTP was bound to the active site, stabilized by interactions with Gly320, Arg323, Lys325, and His329, density for the sugar and base was absent (Fig.…”

“…Efficiency opposite C t decreased by almost four orders of magnitude relative to rGTP insertion in the presence of Mg 2+ . Replacing Mg 2+ with Mn 2+ rescued this decrease for 8-oxo-dGTP insertion 24 , but only partially for the ribonucleotide. The presence of the 2´OH group, in combination with oxidized base damage (Fig.…”

“…Efficiency of undamaged nucleotide insertion was strongly increased opposite both templates, but discrimination remained unaltered. We previously showed 24 that Mn 2+ increases the efficiency of oxidized dGTP (8-oxo-dGTP) insertion opposite C t , but modestly opposite A t , compared to Mg 2+ . Oxidized rGTP (8-oxo-rGTP) was preferentially inserted opposite A t in a single-nucleotide gap, and poorly opposite C t with Mn 2+ .…”

“…While limited information is available on the role and impact of damaged ribonucleotides on DNA replication and repair, insertion of oxidized dGTP (8-oxo-dGTP) by polymerases is known to be mutagenic 22 , 23 . We previously found that 8-oxo-dGTP can base pair with template adenine (A t ) in the pol μ active site upon rotation about the glycosidic bond into the mutagenic syn-conformation 24 . Insertion opposite template cytosine (C t ) in anti-conformation was enabled by active site binding of a third or product metal 25 – 29 , that decreased 8-oxo-dGTP discrimination, such that pol μ efficiently inserted 8-oxo-dGTP opposite either template base.…”