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.

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

“…The minor-groove side of this base pair indicated by the red arrow is solvent-accessible in Dpo4. (BF PDB code: 1LV5, chains A/C/D [11]; Dpo4 PDB code: 2ATL, chains A/D/E [14]. )…”

“…Although the actual dynamic mechanism of translocation is not known for any polymerase, static crystal structures can indicate plausible intermediate states in this process. Recent crystallo-graphic studies with BF [11] and Dpo4 [14] reveal structural rearrangements that accompany dNTP entry into the active site in addition to nucleotide incorporation, thus providing insight into how translocation might occur.…”

“…Dpo4 crystal structures containing a short binary complex, a ternary complex, and a long binary complex following the nucleotidyl-transfer reaction have been investigated recently [14]. Superimposition along their DNA phosphodiester backbones at residues in register revealed no conformational changes akin to the induced-fit motif in high-fidelity DNA polymerases.…”

“…Biochemical processing and X-ray crystal-lographic studies of DNA containing 8-oxoG in complexes with BF [25] and Dpo4 [14,26] have highlighted striking differences in how these polymerases cope with this oxidative guanine lesion. The high-fidelity DNA polymerase BF incorporates the mismatched dATP opposite the damaged base approximately ninefold more efficiently than it does the normal partner dCTP [25], whereas Dpo4 favors dCTP over dATP by > 100-fold [14]. Following incorporation of 2′-deoxycytidine (dC) or 2′-deoxyadenosine (dA), extension occurs much more readily with adenine than with cytosine in BF [25].…”

“…Likewise, X-ray crystallographic structures of Dpo4 in complex with DNA, both with and without dNTP have been determined (for example, see Refs [4,[13][14][15][16][17]). Crystal structures of lesion-containing complexes for these two polymerases are also available (for example, see Refs [14,[18][19][20][21][22][23][24][25][26][27]), which shed light on their contrasting mechanisms for processing damaged DNA templates. We consider two important DNA lesion prototypes as examples of small and bulky lesions, respectively: 7,8-dihydro-8-oxoguanine (8-oxoG; Box 2), a ubiquitous product of reactive oxygen species (ROS), and DNA adducts derived from a tumorigenic metabolite of the environmental pre-carcinogen benzo[a]pyrene (BP; Box 3).…”