DNA mismatch repair (MMR) is a major contributor to genome stability. MMR corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination and is required for the cellular response to certain classes of DNA damage, including lesions induced by several chemotherapeutic drugs (e.g. cisplatin). MMR also has an essential role in somatic hypermutation for the generation of immunoglobin diversity. Defects in human MMR are associated with a strong predisposition to tumor development, and mutagenic expansion of CTG-CAG repeat sequence which are the causative mutations for several meuromuscular diseases (Huntington's disease, fragile-X syndrome, and myotonic dystrophy). Despite the importance of this system in human health and disease, our understanding of its molecular mechanisms is limited. We propose to combine X-ray crystallography, solution small angle X-ray scattering (SAXS) and biochemical approaches to study the human protein-DNA assemblies that are key intermediates in the lesion recognition and excision steps of MMR. We are fortunate that the human MMR pathway has now reached the maturity of analysis that obtaining the protein components and their crystals is now in hand and can be studied directly. We have already determined crystal structures of human MutS? DNA lesion recognition complexes.
In Aim 1 we propose to determine crystal structures of Exo1 and MutS?.
In Aim 2 we investigate substrate recognition, specificity and ATP-dependent conformational transitions of MMR components.
In Aim 3 we study two key multi-component assemblies essential to the recognition and excision process. Together the aims will contribute to furthering our understanding of the molecular basis of MMR-associated human diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Preusch, Peter C
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Duke University
Schools of Medicine
United States
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Shi, Yuqian; Hellinga, Homme W; Beese, Lorena S (2017) Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I. Proc Natl Acad Sci U S A 114:6010-6015
Miller 3rd, Bill R; Beese, Lorena S; Parish, Carol A et al. (2015) The Closing Mechanism of DNA Polymerase I at Atomic Resolution. Structure 23:1609-1620
Wang, Weina; Wu, Eugene Y; Hellinga, Homme W et al. (2012) Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides. J Biol Chem 287:28215-26
Wang, Weina; Hellinga, Homme W; Beese, Lorena S (2011) Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis. Proc Natl Acad Sci U S A 108:17644-8
Tseng, Quincy; Orans, Jillian; Hast, Michael A et al. (2011) Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutS?. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:947-52
Wu, Eugene Y; Beese, Lorena S (2011) The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an ""ajar"" intermediate conformation in the nucleotide selection mechanism. J Biol Chem 286:19758-67
Orans, Jillian; McSweeney, Elizabeth A; Iyer, Ravi R et al. (2011) Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family. Cell 145:212-23