Project Summary/Abstract A fundamental molecular mechanism by which virtually all organisms respond to environmental damage to their genetic material is by carrying out translesion synthesis (TLS) over DNA lesions. The eukaryotic Rev1/3/7-dependent pathway of mutagenic TLS is critically important to human health, not only because it can help cells to survive by tolerating environmental DNA damage, including the repair of DNA crosslinks, but also because this molecular process is responsible for the vast majority of the mutagenesis that occurs as a result of damage to DNA. Mutations from environmental exposure contribute to cancer, other human diseases, and aging. Rev1 is member of the Y family of TLS DNA polymerases, while Rev3 and Rev7 respectively are the catalytic and non-catalytic subunits of TLS DNA pol ?. The overall goal of this research is to build on exciting progress funded by NIEHS grant R01-ES015818 by taking advantage of new developments in human and mammalian biology to gain detailed new insights into the mechanism, regulation, and physiological consequences of this Rev1/3/7-dependent process at a level of resolution that has historically only been attainable using organisms with sophisticated genetic systems such as Escherichia coli and Saccharomyces cerevisiae. A particularly innovative component of this research is to develop a suite of novel inhibitors and other strategies to interfere with Rev1/3/7-dependent TLS, DNA crosslink repair, and other Rev1/3/7-related processes. These will not only be powerful probes to advance basic research into how organisms respond to DNA from environmental chemicals, but also have the potential to improve chemotherapy and possibly other aspects of human health. One major strategy is to identify small molecules that inhibit Rev1/3/7-dependent mutagenic TLS by interfering with critical interactions required for operation of the pathway, such as the interaction of the Rev1 100 amino acid C-terminal domain (CTD) with the Rev7 component of DNA pol ? through one interface and the RIR (Rev1-interacting region) of other TLS DNA polymerases through a second interface. Exemplar compounds have already been identified that bind to each Rev1 Interface and have the expected biological effects. These will be evaluated in syngeic mouse models of human lung cancer and lymphoma as possible chemotherapy adjuvants that increase killing while also reducing the mutagenesis that gives rise to resistance. Other innovative approaches to inhibiting Rev1/3/7-dependent mutagenic TLS include using stapled RIR peptides, using the Anthrax Protective Antigen to deliver the Rev1 CTD into mammalian cells by fusing it to the N-terminus of Lethal Factor, and testing whether Rev7-interacting sequences can serve as dominant negative inhibitors by trapping Rev7 in nonproductive complexes. In a complementary approach, a series of partial-loss-of-function mutants affecting proteins in the Rev1/3/7-dependent-pathway will yield detailed functional insights into the complex protein choreography that underlies the crucial roles of mutagenic TLS in DNA damage tolerance, crosslink repair, and mutagenesis from environmental chemicals.