Cisplatin has been used clinically to treat a variety of cancers for nearly four decades. Along with second and third generation platinum analogs, it is still one of the most widely used chemotherapeutic drugs with over four billion dollars in annual sales. Despite its wide use, clinical limitations including drug toxicity to normal cells and the development of drug resistance in cancers has limited the impact on cancer treatment. Understanding how to overcome these clinical limitations is critical for achieving better cancer responses and increasing overall patient survival. To this end, we have established a novel mechanistic model in which two specific DNA repair pathways, base excision repair (BER) and mismatch repair (MMR), work in conjunction to mediate cisplatin efficacy. This mechanistic model is based on the specific cis/carboplatin DNA interstrand crosslink (ICL) structure in which extrahelical cytosines that flank the ICL are targets for deamination. In this proposal, we have novel preliminary data that supports a family of proteins, APOBEC3 cytidine deaminases, in initiating the deamination of the extrahelical cytosines adjacent to the cisplatin ICLs and activating the BER pathway. Following deamination of the cytosines to uracil, the BER pathway is initiated by uracil DNA glycosylase (UNG) cleavage to produce an abasic site that is subsequently processed by AP endonuclease (APE1) to cleave the phosphodiester backbone adjacent to the cisplatin ICL. Polymerase beta (Polβ) is recruited to the 3'-OH site and synthesizes DNA downstream of the cisplatin ICL, but with poor fidelity. We were the first to demonstrate a dependence on Polβ nucleotide misincorporation to activate MMR, which ultimately inhibits productive ICL DNA repair and maintains cisplatin sensitivity. In our preliminary data, we demonstrate a dependence on APOBEC3 expression to mediate cis/carboplatin sensitivity and activate the BER response. This is further supported by clinical data in which high APOBEC3 expression can mediate a clinical response to cisplatin. We also demonstrate that mutations in Polβ that affect polymerase activity result in hypersensitivity to cisplatin as a consequence of enhanced inhibition of ICL DNA repair. This suggests that the clinically relevant mutations in Polβ, which have been observed in ~30% of tumors tested, that alter polymerase function (e.g., decreased catalytic activity and/or decreased fidelity) may be beneficial for better clinical response to cis/carboplatin treatment as a result of the futile processing of cis/carboplatin ICLs. Here, we propose to (i) elucidate the APOBEC3 family members involved in cisplatin sensitivity and ICL cytosine deamination, (ii) assess the interplay between APOBEC3 members and BER/MMR proteins in cisplatin ICL processing and (iii) identify clinical Polβ mutations that mediate cisplatin efficacy and determine the dependence on APOBEC3 activity. Therefore, this project will provide a comprehensive mechanis...