title: 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.
authors.affiliation: Department of Chemistry and Biochemistry and ‡Science of Advanced Materials Program, Central Michigan University , Mount Pleasant, Michigan 48859, United States.
authors.forename: Wenyan
authors.initials: W
authors.lastname: Xu
authors.forename: Adam M
authors.initials: AM
authors.lastname: Ouellette
authors.forename: Zdzislaw
authors.initials: Z
authors.lastname: Wawrzak
authors.forename: Storm J
authors.initials: SJ
authors.lastname: Shriver
authors.forename: Spencer M
authors.initials: SM
authors.lastname: Anderson
authors.forename: Linlin
authors.initials: L
authors.lastname: Zhao
mesh_terms: D000818:Animals; D019843:Archaeal Proteins / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D058105:Polymerization; D048229:Sulfolobus solfataricus / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D003845:Deoxycytosine Nucleotides; D009711:Nucleotides; C050888:8,5'-cyclo-2'-deoxyguanosine; C024107:2'-deoxycytidine 5'-triphosphate; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine