Stepwise Translocation of Dpo4 Polymerase during Error-Free Bypass of an oxoG Lesion

Received August Stepwise Translocation of Dpo4 Polymerase during Error-Free Bypass of an oxoG Lesion Olga Rechkoblit 0 Lucy Malinina 0 Yuan Cheng 0 Vitaly Kuryavyi 0 Suse Broyde 0 Nicholas E. Geacintov 0 Dinshaw J. Patel 0 Daniel Herschlag, Stanford University, United States of America 0 1 Structural Biology Program, Memorial Sloan-Kettering Cancer Center , New York , New York, United States of America, 2 Biology Department, New York University , New York , New York, United States of America, 3 Chemistry Department, New York University , New York, New York , United States of America 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. - Y-family polymerases are able to bypass a variety of DNA lesions that impede high-fidelity replicative DNA polymerases. Such bypass polymerases exhibit a higher error rate and lower processivity on undamaged DNA templates, and can extend from mismatched base pairs (reviewed in [1,2]). Studies suggest that translesion Y-family DNA polymerases are temporarily recruited to overcome blocks to replicative polymerases [3,4]. Y-family polymerases have more spacious and solvent-accessible active sites as observed for archaeal DNA polymerase IV (Dpo4) [5] and Dbh [6], yeast pol g [7], human pol i [8], and pol j [9], crystallized in the apo form (pol g, Dbh, pol j), and as ternary complexes with an incoming deoxyribonucleotide triphosphate (dNTP) (Dpo4, pol i). The solvent-accessible nature of the active site and the smaller number of contacts of the template-primer DNA with the polymerase enable Dpo4 to accommodate unusual DNA structures in its active site. These include frameshift-template misaligned sequences [5], the cissyn thymine photodimer [10], a bulky benzo[a]pyrene-diol epoxide-adenine lesion [11], an abasic site [12], a reverse wobble G T mismatch [13], and an ethenoguanine lesion [14]. Structural studies have elucidated the effect of metal ions, nucleotide selection, and pyrophosphorolysis on Dpo4 fidelity [15]. By contrast, replicative polymerases produce tight-fitting, solvent-excluding active sites upon binding of the correct nucleotide; the O helix in the finger domain undergoes a large movement to position itself on the flat surface of a complementary, Watson-Crick nascent base pair [3,16]. This may represent a kinetic ratelimiting step that occurs prior to covalent nucleotide incorporation which has been interpreted in terms of an induced-fit mechanism [17]. If an unusual DNA alignment or a damaged base is encountered at the active site, the O helix often remains in the open, inactive conformation [1821]. In striking contrast to the replicative polymerases, the finger domains of Yfamily Dpo4, Dbh, pol g, pol i, and pol j polymerases are missing the equivalent of the O helix [8,9,22]. Instead, the replicating base pair is contacted by a b-sheet (Dpo4 residues 4146) of the finger domain, which forms the rigid roof of the active site, and by an adjacent extended loop (Dpo4 residues 5359). Kinetic studies indicate that the Yfamily pol g and Dpo4 employ a rate-limiting protein conformational change before covalent nucleotide incorporation occurs at the active site [23,24]. However, examinations of currently available crystal structures of Y-family polymerases do not reveal any obvious conformational changes, and it has been suggested that bypass polymerases, such as Dpo4, are always in the closed, ready-for-catalysis conformation [22,25]. The absence of an open-to-closed conformational transition was also observed in the case of the repair gapfilling pol k [26]; the dNTP is accommodated in the space that was, in the binary complex, occupied by the side chain of a tyrosine residue, and the template strand is repositioned toward the active site. 7,8-dihydro-8-oxoguanine (oxoG) is the major known product of oxidation of DNA by reactive oxygen species induced by either ionizing radiation, photochemical mechanisms, or normal cellular metabolic activity [27]. An increased risk for developing cancer has been linked to oxidative stress due to the overproduction of reactive oxygen species resulting from the response of cells to inflammation and infection [28]. Replicative polymerases in vitro readily insert C or A opposite the oxoG lesion in varying proportions that depend on the polymerase, with extension occurring preferentially from oxoG A mispairs [2931]. In contrast, the Yfamily polymerases yeast and human pol g [32], and Dpo4 (this study), preferentially insert C opposite oxoG, and also preferentially extend from the oxoG C base pair, thus achieving error-free bypass of this lesion. To date, a number of crystal structures have been reported for replicative polymerases with oxoG-modified templateprimers and dNTPs positioned opposite the lesion or the adjacent 59-template base, corresponding to insertion and extension ternary complexes, respectively. These include insertion ternary complexes, with oxoG positioned opposite 29-deoxycytidine 59-triphosphate (dCTP) and 29-deoxyadenosine 59-triphosphate (dATP), with the repair gapfilling pol b [33], and opposite dCTP for the replicative polymerases Rb69 [18] and pol T7 [34], as well as extension complexes past oxoG C and oxoG A base pairs by pol T7 [34] and Bacillus pol I [35]. The objectives of this study are to address a major gap in our understanding of the conformational changes associated with the polymerase translocation steps that occur in Y-family polymeraseDNA substrate complexes that accompany dNTP insertion and the nucleotidyl transfer reaction. We have chosen Dpo4 due to its high homology (...truncated)