Free radical damage to DNA encompasses an array of oxidized bases and abasic lesions with fragmented or oxidized deoxyribose. Among these, 2-deoxyribonolactone (dL) presents a special problem: attempted processing of dL via the mammalian single-nucleotide base excision repair pathway can generate irreversible crosslinks with DNA polymerase β (Polβ). We have demonstrated that these DNA-protein crosslinks (DPC) are formed in vivo in a variety of human and mouse cell lines, most effectively by agents that generate the most dL. This DPC formation depends on the Polβ active site for its 5’-lyase activity that excises normal deoxyribose-5-phosphate and is specific to oxidative agents. Rapid proteolytic processing of the Polβ-DPC removes most of the protein within ~30 minutes, and we have identified some of the key early steps in this process. Other DNA repair proteins with abasic lyase activity also form DPC in cells treated with dL-forming oxidants, including the end-binding Ku proteins, which have a pattern of trapping similar to that of Polβ. The Ku-DPC are also rapidly processed following their formation.  We have also detected in vivo DPC formation with five other repair enzymes that display unique reaction patterns, and these studies will also be presented.

Enzyme mechanism-driven DNA-protein crosslinks during attempted DNA repair

253rd National Meeting (2017)

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