Probing the DNA-binding center of the MutL protein from the Escherichia coli mismatch repair system via crosslinking and Förster resonance energy transfer

Monakhova, M and Ryazanova, A and Kunetsky, V and Li, P and Shilkin, E and Kisil, O and Rao, DN and Oretskaya, T and Friedhoff, P and Kubareva, E (2020) Probing the DNA-binding center of the MutL protein from the Escherichia coli mismatch repair system via crosslinking and Förster resonance energy transfer. In: Biochimie, 171-72 . pp. 43-54.

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Abstract

As no crystal structure of full-size MutL bound to DNA has been obtained up to date, in the present work we used crosslinking and Förster resonance energy transfer (FRET) assays for probing the putative DNA-binding center of MutL from Escherichia coli. Several single-cysteine MutL variants (scMutL) were used for site-specific crosslinking or fluorophore modification. The crosslinking efficiency between scMutL proteins and mismatched DNA modified with thiol-reactive probes correlated with the distances from the Cys residues to the DNA calculated from a model of MutS�MutL�DNA complex. FRET-based investigation of DNA binding with different scMutL variants clearly showed that the highest signals were detected for the variants MutL(T218C) and MutL(A251C) indicating closeness of the positions 218 and 251 to DNA in the MutL�DNA complex. Indeed, the Cys218 and Cys251 of scMutL were crosslinked to the reactive DNA with the highest yield demonstrating their proximity to DNA in the MutL�DNA complex. The presence of MutS increased the yield of conjugate formation between the MutL variants and the modified DNA due to tighter MutL�DNA interactions caused by MutS binding to MutL. © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)

Item Type:	Journal Article
Publication:	Biochimie
Publisher:	Elsevier B.V.
Additional Information:	The copyright of this article belongs to Elsevier B.V.
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	27 Jul 2021 10:28
Last Modified:	27 Jul 2021 10:28
URI:	http://eprints.iisc.ac.in/id/eprint/64798

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