PROJECT SUMMARY DNA crosslinks, including interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs) are forms of DNA damage that arise continuously in DNA from endogenous and natural sources. They must be removed in order to allow accurate genome duplication and gene expression. Despite the importance of crosslinks in cancer etiology and treatment, mechanisms of DNA crosslink repair are known only in outline, and many steps are simply assumed. Project 1 is concerned with the biochemistry of key steps at the heart of the mechanism of ICL repair. When DNA replication fork stalling occurs at an ICL, a frequent outcome is processing by structure- specific DNA nucleases to unhook the crosslink. ERCC1-XPF is implicated as a key enzyme for ICL unhooking. Major unknowns include the nature of the substrates that are cut, and the roles of other components that are involved in ICL unhooking. ERCC1-XPF interacts with SLX4-SLX4IP, MSH2-MSH3, and RPA and is stimulated by these components, but their roles are not understood. In Aim 1, research will define conditions under which ERCC1-XPF efficiently cleaves an ICL fork structure in concert with a protein complex containing SLX4, MSH2-MSH3, and DNA binding proteins. The mechanism of another critical step, the bypass of the unhooked ICL, will be assessed in Aim 2. Major unknowns are whether the pol ζ holoenzyme can fully bypass an unhooked ICL, if all subunits are necessary, and whether an additional DNA polymerase is required. The contribution of other DNA polymerases to ICL processing will be assessed systematically in defined cellular assays. We will determine whether single-stranded DNA binding proteins including HMGB1/2/3 promote bypass. We will biochemically determine subunits and activities required for unhooked ICL bypass using a newly developed expression and purification system for pol ζ. Bypass of differently sized DNA-peptide crosslinks will be examined with the same group of DNA polymerases. We will also examine the mechanism of recruitment of pol ζ to crosslinked damaged DNA by newly discovered interfaces with nuclease components. The Central Hypothesis of Project 1 is that is that many under-studied protein factors that have major influences on ICL cleavage and in bypass of crosslinked structures. These studies will address overall Program Project Goals defining Pathways of crosslink repair, new Components, and repair of DNA-Protein crosslinks.