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.

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 published 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. under review.

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. in press.

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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.

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. In the longer term, this research will be extended to other families of lesions and additional bypass polymerases.