SUMMARY - PROJECT 1: Molecular response to base-modifying chemotherapeutic agents Repair of base damage on DNA is critical because such damage is both genotoxic and mutagenic. Many chemotherapeutic agents used for cancer therapy, such as cisplatin and alkylating agents, induce this type of damage and are one of the most commonly used modalities for treatment, underscoring their clinical significance. While much is known about how cells respond to DNA containing base damage, the importance of RNA damage has become increasingly appreciated only recently. Indeed, alkylated RNA is estimated to comprise the vast majority of the damaged nucleic acid in cells treated with such agents. It is not surprising then that cells have evolved a number of mechanisms to cope with damaged RNA. We have discovered that the ALKBH3-ASCC repair complex mediates the cellular response to DNA as well RNA base damage. This complex also interfaces with the innate immune or DAMP (damage-associated molecular pattern) signaling through a novel RNA endonuclease activity, which we have recently uncovered through the multidisciplinary efforts of this program project. Our work implicates RNA damage recognition and signaling as a critical node in the cellular response to base damage, an underexplored area, which we seek to understand in this renewal application. Specifically, we will use a combination of structural methods and biochemistry to reveal the mechanistic basis of the enzymatic activities within the ALKBH3-ASCC complex (Aim 1). We will also determine the importance of ASCC-mediated, RNA-dependent DAMP signaling in the response to commonly used chemotherapeutic agents and its role in cell fate decisions (Aim 2). Finally, we will determine how the ALKBH3-ASCC pathway integrates with established base repair pathways, potentially identifying novel additive or synthetic lethal relationships between these pathways (Aim 3). Together, the proposed work will reveal important insights into the contribution of RNA as well as DNA base damage responses and its relevance to tumor cell eradication.