Article

In vitro studies of DNA mismatch repair proteins.

Genetics & Biochemistry Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Analytical Biochemistry (Impact Factor: 2.31). 02/2011; 413(2):179-84. DOI: 10.1016/j.ab.2011.02.017
Source: PubMed

ABSTRACT The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O(6)meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.

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Available from: Chunwei Du, Sep 18, 2014
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    • "Using such cheap, commercially available gel extraction kits is likely the fastest (30 min–2 h) and easiest approach for DNA purification. Alternatively, DNA substrate extraction can also be achieved from solution via phenol/chloroform or via a CsCl density gradient, as described elsewhere (Hou et al., 2007; Geng et al., 2011; Ristic et al., 2011 "
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    ABSTRACT: Protein-DNA interactions provide fundamental control mechanisms over biologically essential processes such as DNA replication, transcription, and repair. However, many details of these mechanisms still remain unclear. Atomic force microscopy (AFM) analyses provide unique and important structural and functional information on such protein-DNA interactions at the level of the individual molecules. The high sensitivity of the method with topographical visualization of all sample components also demands for extremely clean and pure materials. Here, we provide an overview of molecular biology-based approaches to produce DNA substrates for AFM imaging as well as other types of experiments, such as optical or magnetic tweezers, that profit from controllable substrate properties in long DNA fragments. We present detailed strategies to produce different types of motifs in DNA that are frequently employed targets of protein interactions. Importantly, the presented preparation techniques imply exact knowledge of the location of the introduced specific target sites within the DNA fragments, allowing for a distinction between specific and non-specific protein-DNA interactions in the AFM images and for separate conformational analyses of the different types of protein-DNA complexes. Copyright © 2013 John Wiley & Sons, Ltd.
    Journal of Molecular Recognition 12/2013; 26(12):605-17. DOI:10.1002/jmr.2311 · 2.34 Impact Factor
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    • "Using such cheap, commercially available gel extraction kits is likely the fastest (30 min–2 h) and easiest approach for DNA purification. Alternatively, DNA substrate extraction can also be achieved from solution via phenol/chloroform or via a CsCl density gradient, as described elsewhere (Hou et al., 2007; Geng et al., 2011; Ristic et al., 2011 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein–DNA interactions provide fundamental control mechanisms over biologically essential processes such as DNA replication, transcription, and repair. However, many details of these mechanisms still remain unclear. Atomic force microscopy (AFM) analyses provide unique and important structural and functional information on such protein–DNA interactions at the level of the individual molecules. The high sensitivity of the method with topographical visualization of all sample components also demands for extremely clean and pure materials. Here, we provide an overview of molecular biology‐based approaches to produce DNA substrates for AFM imaging as well as other types of experiments, such as optical or magnetic tweezers, that profit from controllable substrate properties in long DNA fragments. We present detailed strategies to produce different types of motifs in DNA that are frequently employed targets of protein interactions. Importantly, the presented preparation techniques imply exact knowledge of the location of the introduced specific target sites within the DNA fragments, allowing for a distinction between specific and non‐specific protein–DNA interactions in the AFM images and for separate conformational analyses of the different types of protein–DNA complexes. Copyright © 2013 John Wiley & Sons, Ltd.
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    ABSTRACT: The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.
    Journal of Biological Chemistry 01/2012; 287(13):9777-91. DOI:10.1074/jbc.M111.316919 · 4.60 Impact Factor
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