MISMATCH REPAIR AND CARCINOGENESIS PROJECT SUMMARY / ABSTRACT Defects in human mismatch repair (MMR) are the cause of Lynch syndrome aka hereditary non-polyposis colorectal cancer (LS/HNPCC), as well as 10-40% of various sporadic cancers. MMR corrects DNA polymerase misincorporation errors, suppresses recombination between non-allelic partially homologous DNA sequences, and functions as a lesion sensor in DNA damage signaling. The unrepaired errors in MMR-deficient cells lead to increased genomic mutations that drives tumorigenesis, while the lack of damage sensing results in resistance to several common chemotherapeutics. Conversely, MMR defective tumors appear to activate cellular innate immunity that results in strikingly effective PD1/PD-L1-based immunotherapy. Despite decades of study, critical aspects of MMR mechanics remain uncertain, the sequence of events that leads to MMR damage signaling is poorly understood, and the mechanism that connects MMR to innate immunity is largely a mystery. Four MMR genes account for most of the LS/HNPCC mutations and include the highly conserved MutS homologs (MSH) and MutL homologs (MLH/PMS) MSH2, MSH6, MLH1 and PMS2. During the last funding period, we expanded our single molecule imaging capabilities and discovered that the MSH and MLH/PMS proteins interact to form novel cascading sliding clamps on a mismatched DNA. Once loaded by an MSH, the MLH/PMS sliding clamps assist in detecting the error-containing strand and function as a processivity factor for MMR excision activities, while retaining a large enough “donut hole” to transit nucleosomes. The remarkable stability and DNA diffusion properties of the MSH and MLH/PMS sliding clamps clarified our understanding of MMR mechanisms, while provoking new concepts for strand-specific excision and damage signaling. Remarkably, the MSH and MLH/PMS sliding clamps displayed entirely random motions when on the DNA. Deterministic mechanisms have historically underpinned MMR models, where static complexes and well-defined stepwise biochemical sequences are proposed to complete repair events. This renewal application will explore the hypothesis that the entire multi-component multi-pathway MMR process is Stochastic: governed by chance encounters, varied repair intermediates and/or MMR component exchanges that are orchestrated and anchored to the DNA by dynamic MSH and MLH/PMS sliding clamps. Understanding the depth of biochemical randomness during MMR will facilitate similar biophysical studies of more complex DNA repair systems. We propose the following Specific Aims: 1) examine the interactions and competition between human MMR components in real-time, 2) detail the dynamic selection and interchange between strand-specific excision components during MMR, 3) examine fundamental damage recognition and signaling interactions on chromatin substrates, and 4) visualize human MMR component interactions with single molecule resolution in vivo. The overall goal of this resea...