Defects in the human mismatch repair (MMR) genes are the cause of Lynch syndrome (LS/HNPCC), as well as 10-40% of sporadic colorectal, gastric, endometrial, ovarian and upper urinary tract tumors. MMR recognizes and repairs polymerase misincorporation errors, suppresses recombination between non-allelic partially homologous DNA sequences, and functions as a lesion sensor in DNA damage signaling. Unrepaired errors in MMR-deficient cells lead to increased mutations (Mutator Phenotype) that drive tumorigenesis and MMR-deficient tumors are resistant to several common cancer chemotherapeutic drugs. MMR is a bidirectional excision-resynthesis process that is initiated at a distant DNA strand break, which may be located 3' or 5' and hundreds to thousands of nucleotides from the mismatch. The fidelity of post-replication MMR depends on communicating mismatch recognition to the distant strand break and then exclusively directing excision to the DNA strand containing the misincorporation error. Resynthesis of the resulting DNA gap is independent from MMR-directed excision and performed by the replicative polymerase. How MMR components coordinate mismatch or lesion recognition with the numerous downstream activities that perform DNA excision or damage signaling remains uncertain. In the last 5-year period of support we developed unique real-time single molecule imaging methods that visualized fundamental protein functions and detailed the ensemble 5'-MMR excision process. The most intriguing observation was that every step of MMR appeared stochastic. Dynamic often non-productive MMR complexes undergoing thermal (Brownian) motion on the mismatched DNA were continuously formed and dissolved during the excision process. While these superficially random progressions showcased the chaotic nature of biology, it was apparent that sufficient productive events occurred to faithfully complete MMR in most cases. In this renewal application we propose to detail how these dynamic processes are coordinated in vitro and in vivo to clearly determine the mechanism of MMR. It is our hypothesis that many of the several hundred LS/HNPCC missense mutations affect poorly understood stochastic steps in MMR and DNA damage signaling. We will accomplish the following Specific Aims: 1.) visualize the complete ensemble human 3'- and 5'-MMR process in vitro in single molecule detail, 2.) examine the dynamic interactions between defined physiologically relevant chromatin and human MMR, and 3.) examine human MMR component interactions with single molecule detail in vivo. The goal of this proposal is to visualize and quantify the MMR progressions that ultimately lead to cancer and therapeutic drug resistance.
More than 700 unique genetic alterations of the human mismatch repair (MMR) genes have been reported in families susceptible to LS/HNPCC (http://www.hgmd.cf.ac.uk), of which 20-75% are missense alterations with unknown functional consequences. This proposal will develop and utilize innovative single molecule imaging and analytical methods capable of visualizing and quantitatively examining the ensemble human MMR. These studies will provide a platform for understanding the detailed function(s) of the core MMR components in excision repair, damage signaling, drug resistance and tumorigenesis
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| Martín-López, Juana; Gasparini, Pierluigi; Coombes, Kevin et al. (2018) Mutation of TGF?-RII eliminates NSAID cancer chemoprevention. Oncotarget 9:12554-12561 |
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| Fishel, Richard; Heinen, Christopher D (2016) Enhanced gene targeting to evaluate Lynch syndrome alterations. Proc Natl Acad Sci U S A 113:3918-20 |
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| Lee, Jong-Bong; Cho, Won-Ki; Park, Jonghyun et al. (2014) Single-molecule views of MutS on mismatched DNA. DNA Repair (Amst) 20:82-93 |
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