BYPASS FIDELITY OF OXIDATIVE DAMAGE LESIONS BY Y-FAMILY DNA POLYMERASE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Genomic integrity depends critically on the fidelity and efficiency of DNA replication. High-fidelity DNA polymerases that replicate genomic DNA can stall on certain DNA damage sites, and one or more lesion-bypass Y-family polymerases are recruited to transit the lesion. Such bypass polymerases have a higher error rate and lower processivity on undamaged DNA templates, but can insert a base opposite a lesion site and extend from a damaged base pair in error-free (mutation-avoiding) or error-prone (mutation-generating) manner. Our goal is to understand the molecular mechanisms that define the mutagenic events associated with replication of oxidative damage lesions by bypass polymerases. 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. Elevated levels of oxidative damage lesions in genomic DNA have also been associated with neurodegenerative diseases, aging, and cardiovascular disorders. Our crystallographic studies are undertaken on the most prevalent oxidative damage lesions, namely, 8-oxoguanine, the stable ring-opened 5-guanidino-4-nitroimidazole adduct and the fused bicyclic spiroiminodihydantoin adduct, placed in the context of DNA template strands of the active site of Dpo4 Y-family polymerase. Previously, our group uncovered structural and functional features that enable low-fidelity Dpo4 polymerase to achieve predominantly error-free insertion of a cytosine base opposite the 8-oxoguanine (oxoG) lesion. We found of The oxoG lesion adopts an anti conformation within the Dpo4 active site, that is necessary for pairing with dCTP, with the recognition event facilitated by multiple and favorable contacts of Dpo4 amino acid residues with the oxoG. We have also demonstrated that Dpo4 undergoes stepwise translocations throughout the catalytic cycle that are distinct from the corresponding translocation events observed in high-fidelity polymerases. Our efforts should elucidate the common factors that promote error-free or error-prone DNA synthesis opposite and past the oxidative damage lesions of varying size and shape, as well as lesion architecture and preferred conformations.