title: Stepwise translocation of Dpo4 polymerase during error-free bypass of an oxoG lesion.
abstract: 7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG*C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain-DNA phosphate contacts translocate by one nucleotide step, while the thumb domain-DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain-phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.
authors.affiliation: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Rechkoblit
authors.forename: Lucy
authors.initials: L
authors.lastname: Malinina
authors.forename: Yuan
authors.initials: Y
authors.lastname: Cheng
authors.forename: Vitaly
authors.initials: V
authors.lastname: Kuryavyi
authors.forename: Suse
authors.initials: S
authors.lastname: Broyde
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Geacintov
authors.forename: Dinshaw J
authors.initials: DJ
authors.lastname: Patel
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D019951:DNA Polymerase beta / Q000235:genetics* / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000502:physiology; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000502:physiology; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D048229:Sulfolobus solfataricus / Q000201:enzymology; D013698:Templates, Genetic; D014178:Translocation, Genetic
chemical_list: C453560:7,8-dihydro-8-oxoguanine; D019843:Archaeal Proteins; D003845:Deoxycytosine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D006147:Guanine; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase