Kinetic and Structural Mechanisms of (5′S)-8,5′-Cyclo-2′-deoxyguanosine-Induced DNA Replication Stalling

Abstract

The (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) lesion is produced from reactions of DNA with hydroxyl radicals generated from ionizing radiation or endogenous oxidative metabolisms. An elevated level of S-cdG has been detected in Xeroderma pigmentosum, Cockayne syndrome, breast cancer patients, and aged mice. S-dG blocks DNA replication and transcription in vitro and in human cells and produces mutant replication and transcription products in vitro and in vivo. Major cellular protection against S-dG includes nucleotide excision repair and translesion DNA synthesis. We used kinetic and crystallographic approaches to elucidate the molecular mechanisms of S-cdG-induced DNA replication stalling using model B-family Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) and Y-family S. solfataricus P2 DNA polymerase IV (Dpo4). Dpo1 and Dpo4 inefficiently bypassed S-cdG with dCTP preferably incorporated and dTTP (for Dpo4) or dATP (for Dpo1) misincorporated. Pre-steady-state kinetics and crystallographic data mechanistically explained the low-efficiency bypass. For Dpo1, S-cdG attenuated Kd,dNTP,app and kpol. For Dpo4, the S-cdG-adducted duplex caused a 6-fold decrease in Dpo4:DNA binding affinity and significantly reduced the concentration of the productive Dpo4:DNA:dCTP complex. Consistent with the inefficient bypass, crystal structures of Dpo4:DNA(S-cdG):dCTP (error-free) and Dpo4:DNA(S-cdG):dTTP (error-prone) complexes were catalytically incompetent. In the Dpo4:DNA(S-cdG):dTTP structure, S-cdG induced a loop structure and caused an unusual 5'-template base clustering at the active site, providing the first structural evidence of the previously suggested template loop structure that can be induced by a cyclopurine lesion. Together, our results provided mechanistic insights into S-cdG-induced DNA replication stalling.

Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ

“…Primer-extension assays were performed and analyzed as previously described. 28 Reaction conditions are specified as follows. Full-length extension assays were performed at 37 °C using 80 nM primer-template DNA, 80 nM DNA polymerase, four dNTPs at their physiological concentrations 29 (i.e., 10 μ M for dGTP and 40 μ M for dATP, dCTP, and dTTP), 4% (v/v) glycerol, 5 mM DTT, 50 mM NaCl, 5 mM MgCl 2 , and 100 μ g/mL bovine serum albumin (BSA) in 50 mM Tris-HCl (pH 7.4 at 25 °C).…”

Mutagenic Bypass of an Oxidized Abasic Lesion-Induced DNA Interstrand Cross-Link Analogue by Human Translesion Synthesis DNA Polymerases

“…Assays were performed according to a published procedure. 38 Specifically, full-length primer extension experiments were conducted at 37 °C with 80 nM duplex DNA (with or without ICL), 80 nM DNA polymerase, four dNTPs at 100 μ M each, 4% (v/v) glycerol, 5 mM DTT, 50 mM NaCl, 5 mM MgCl 2 , and 100 μ g mL −1 bovine serum albumin (BSA) in 50 mM Tris-HCl (pH 7.4 at 25 °C). Assays with pol ζ contained 10% glycerol and 90 mM KCl.…”

O6-2′-Deoxyguanosine-butylene-O6-2′-deoxyguanosine DNA Interstrand Cross-Links Are Replication-Blocking and Mutagenic DNA Lesions

“…The observed partially extended products in gel electrophoretic analysis suggest the potential for truncation or deletion mutations (likely due to looping-out the lesion during catalysis, see refs 26 and 37 for examples); however, the sequence of these products remains obscure. To address this question, we used LC-MS-based oligonucleotide sequencing to identify the bypass products as well as to determine their relative yields.…”

“…Assays were performed according to published procedures. 26 Briefly, full-length primer-extension experiments were conducted at 37 °C with 100 nM duplex DNA (with or without ICL), 80 nM DNA pol, four dNTPs at 100 μ M each, 4% (v/v) glycerol, 5 mM DTT, 50 mM NaCl, 5 mM MgCl 2 , and 100 μ g mL −1 bovine serum albumin (BSA) in 50 mM Tris-HCl (pH 7.4 at 37 °C). Extension assays with cell extracts were performed with an ICL-S-containing duplex.…”