Replication of DNA Damage Sites by Bypass Polymerases

Oxidative Lesions

Reactive electrophilic oxygen species, the byproducts of aerobic respiration, can damage DNA, contributing to mutagenesis and carcinogenesis. These events are accelerated under conditions of oxidative stress, such as during inhalation or exposure to cigarette smoke. We are undertaking crystallographic studies on the most prevalent oxidative damage lesions, namely 8-oxoguanine, the stable ring-opened 5-guanidino-4-nitroimidazole adduct and the bulky fused spiroiminodihydantoin adduct, positioned at template-primer junctions, as part of preinsertion and postinsertion binary and ternary (with incoming nucleoside triphosphate) complexes, with the thermophilic Dpo4 bypass polymerase.

Our efforts should elucidate the geometric fit, alignment and register for individual oxidative damage lesions of varying size and shape positioned in the active site of Dpo4, should determine the specific interactions and pairings of the lesion site with complementary and non-cognate incoming nucleoside triphosphates, and should identify key residues and alignments for facilitating the divalent cation-mediated nucleotidyl transfer reaction. The proposed studies should provide structural insights into how bypass is modulated by lesion architecture and base sequence context, and provide explanations for the distribution of point mutations relative to frame-shift deletions.

This research involves a long-term collaboration on the structure and processing of DNA lesions with the Nicholas Geacintov and Suse Broyde laboratories at New York University and the John Essigmann laboratory at MIT.

Our laboratory has contributed to the following reviews on bypass polymerases:

Broyde, S., Wang, L., Rechkoblit, O., Geacintov, N. E. & Patel, D. J. (2008). Lesion processing by replicative versus bypass polymerases. Trends Biochem. Scis. 33, 209-219.

Broyde, S., Wang, L., Zhang, L., Rechkoblit, O., Geacintov, N. E. & Patel, D. J. (2008). DNA adduct structure-function relationships: comparing solution with polymerase structure. Chem. Res. Toxicol. 21, 45-52.

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We have already solved the structures of both binary and ternary complexes that have provided detailed insights into dCTP-binding and dCTP-incorporation steps, and in the longer term, elongation steps. These structures have provided insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during non-covalent dCTP insertion opposite oxoG the little finger domain-DNA phosphate contacts translocate by one nucleotide step, while the thumb domain contacts with DNA phosphates 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.

Rechkoblit, O., Malinina, L., Cheng, Y., Kuryavyi, V., Broyde, S., Geacintov, N. & Patel, D. J. (2006). Stepwise translocation of Dpo4 polymerase during error-free bypass of oxoG lesion. PLoS Biology 4, 25-42.

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7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA, is processed differently by high-fidelity and Y-family lesion bypass polymerases. While high-fidelity polymerases extend predominantly from an A base opposite an oxoG, the Y-family polymerases Dpo4 and human pol eta preferentially extend from the oxoG·C base pair. We have determined crystal structures of extension Dpo4 ternary complexes with oxoG opposite C, A, G, or T and the next correct nascent base pair. We demonstrate that neither template backbone nor the architecture of the Dpo4 active site is perturbed by either the oxoG(anti)·C or the oxoG·A pairs; however, the latter manifest conformational heterogeneity, adopting both oxoG(syn)·A(anti) and a novel oxoG(anti)·A(syn) alignment; Arg332 from the little finger domain governs the anti-syn equilibrium of the oxoG residue. The anti-syn equilibrium of the oxoG residue, in turn, triggers the syn-anti equilibrium of partner base A, and, thus, reduces extension from the dynamically flexible 3'-terminal primer base A. Because of structural and functional homologies between Dpo4 and pol eta, such a dynamic screening mechanism might also be utilized for proof-reading by pol eta during error-free bypass of oxoG and, in general, by Dpo4 and pol eta to regulate error-free versus error-prone nucleotide incorporation for other lesions.

Rechoblit, O., Malinina, L., Cheng, Y., Geacintov, N. E., Broyde, S. & Patel, D. J. (2009). Impact of conformational heterogeneity of oxoG lesion and its pairing partners on bypass fidelity by Y family polymerases. Structure 17, 633-634.