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Time: 4:00 pm-5:00 pm
Dr. Michael B. Smeaton, of Stanford University, presents "Interstrand cross-link repair in mammals involves TopoisomeraseIIβ".
Interstrand cross-links (ICLs) are a type of DNA damage that covalently tether the opposing strands of the DNA helix. ICLs are formed by some of the most commonly used chemotherapeutics, repair of which can result in resistance. ICLs can also form through endogenous mechanisms and, if left unrepaired, can contribute towards aging processes.
The pathways that repair ICLs are diverse, however, the proteins that initiate the repair process remain unknown. Here, we examine pathways of ICL repair available to terminally differentiated cells and the proteins that initiate the repair process. Model, site-specific ICLs were synthesized that either fit perfectly into B-form DNA or cause a high level of helical distortion.
Using mammalian cell extracts, a biochemical activity was uncovered that ‘unhooks’ the ICL, the critical first step in the repair process. The unhooking reaction was damage-specific, ATP/Mg2+ dependent and responded to the levels of helical distortion.
In yeast and bacteria, the nucleotide excision repair (NER) pathway is responsible for the initial unhooking step in ICL repair. However, we found the unhooking activity to be independent of the NER pathway in mammalian cells.
Purification of the proteins responsible for unhooking was achieved through fractionation of cell extracts. Mass spec analysis of the final, active fraction identified topoisomerase II. Humans have two isoforms of topoII, α and β. The topoII catalytic inhibitor, merbarone, was able to abolish the unhooking activity in extracts. Recombinant topoIIβ, but not α, was able to nucleolytically process a purified ICL substrate.
Finally, using a plasmid based host-cell reactivation assay, we
confirm the involvement of topoIIβ in repair of ICLs in vivo. These findings have significant clinical implications in treating chemoresistant tumors with readily available drugs.
Room: Science Building B - Room 133
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