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MutS recognition: Multiple mismatches and sequence context effects

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Abstract

Escherichia coli MutS is a versatile repair protein that specifically recognizes not only various types of mismatches but also single stranded loops of up to 4 nucleotides in length. Specific binding, followed by the next step of tracking the DNA helix that locates hemi-methylated sites, is regulated by the conformational state of the protein as a function of ATP binding/hydrolysis. Here, we study how various molecular determinants of a heteroduplex regulate mismatch recognition by MutS, the critical first step of mismatch repair. Using classical DNase I footprinting assays, we demonstrate that the hierarchy of MutS binding to various types of mismatches is identical whether the mismatches are present singly or in multiples. Moreover, this unique hierarchy is indifferent both to the differential level of DNA helical flexibility and to the unpaired status of the mismatched bases in a heteroduplex. Surprisingly, multiple mismatches exhibit reduced affinity of binding to MutS, compared to that of a similar single mismatch. Such a reduction in the affinity might be due to sequence context effects, which we established more directly by studying two identical single mismatches in an altered sequence background. A mismatch, upon simply being flipped at the same location, elicits changes in MutS specific contacts, thereby underscoring the importance of sequence context in modulating MutS binding to mismatches.

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Abbreviations

DEPC:

Diethyl pyrocarbonate

DTT:

dithiothreitol

IDLs:

insertion deletion loops

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Authors and Affiliations

  1. Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, 400 005, Mumbai, India

    Amita Joshi & Basuthkar J. Rao

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  1. Amita Joshi
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  2. Basuthkar J. Rao
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Correspondence to Basuthkar J. Rao.

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Joshi, A., Rao, B.J. MutS recognition: Multiple mismatches and sequence context effects. J. Biosci. 26, 595–606 (2001). https://doi.org/10.1007/BF02704758

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