Structural and functional analysis of the MutS C-terminal tetramerization domain

Manelytė, Laura; Urbanke, Claus; Giron-Monzon, Luis; Friedhoff, Peter

doi:10.1093/nar/gkl489

Cited by 32 publications

(55 citation statements)

References 33 publications

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“…S6). Moreover, we repeated the experiments with doubly labeled substrates with the MutSΔ800 mutant protein, which lacks the C-terminal domain required for tetramerization, and the MutS-R840E mutant protein, which disrupts tetramerization (36,37). The CSFs from the modified protein were identical to within noise for each condition.…”

Section: Resultsmentioning

confidence: 99%

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

Hura

Tsai

Claridge

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flashcooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. D NA is frequently considered a passive component in interactions with proteins involved in DNA metabolism. Despite this view, many proteins use DNA structural features to mediate catalysis and identify damaged DNA through the effects of damage on DNA rigidity and conformation (1-6). The view of DNA as a passive element is therefore at least in part due to a paucity of robust tools to examine dynamic DNA conformational states during multistep reactions. Gold-labeled DNA enables measurement over length scales sufficient to accommodate several proteins to identify cooperative effects on DNA. Because X-rays scatter predominantly from electrons, using heavy atom labels (7-11) provides high contrast relative to organic molecules. By using labels of moderate size (∼5 nm), the scattering from gold nanocrystals dominates all other scattering signals by three orders of magnitude, thereby reducing analysis complexity while minimizing nanocrystal influence on biological macromolecules. Importantly, small angle X-ray scattering (SAXS) provides global information on conformations adopted by a population of macromolecules in almost any solution condition (12)(13)(14).Mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication (15,16). Despite the i...

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Section: Resultsmentioning

confidence: 99%

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

Hura

Tsai

Claridge

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Site-directed Mutagenesis-Plasmids encoding the gene for a cysteine-free MutS (MutS-CF) and dimeric MutS-CF/D835R have been described before (8). Single cysteine MutS variants (Table 1 and supplemental Table S1) were generated by sitedirected mutagenesis using a modification of the QuikChange protocol (35,36).…”

Section: Methodsmentioning

confidence: 99%

“…Bacterial MutS exists in a dimer-tetramer equilibrium, but the dimeric form of the protein is sufficient for DNA mismatch repair (8,9). MutS is composed of seven domains (mismatchbinding, connector, core, lever, clamp, ATPase, and dimerization/tetramerization domains) (see Fig.…”

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confidence: 99%

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli

Winkler

Marx

Larivière³

et al. 2011

Journal of Biological Chemistry

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“…Complexes were resolved on a 6% acrylamide Tris borate-EDTA non-denaturing gel (Invitrogen) after a 30-min migration at 22°C, visualized, and quantified by phosphorimaging. The best fit of titration curves were generated with a cooperative binding model using the following sigmoidal equation, as previously described (20)…”

Section: Methodsmentioning

confidence: 99%

Essential Role of the N-terminal Domain in the Regulation of RIG-I ATPase Activity

Gee

Chua

Gevorkyan

et al. 2008

Journal of Biological Chemistry

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Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-␤ expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.Pathogen recognition receptors constitute one of the first lines of human host cell defense against viral infections. Among them, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) helicases are specialized sensors of viral nucleic acid. RIG-I was shown to be involved in the recognition of Sendai virus, Hepatitis C virus (HCV), 2 and vesicular stomatitis virus, whereas MDA-5 mediates the antiviral response to picornaviruses (1-3). Both RIG-I and MDA-5 are modular proteins of ϳ920 residues that contain an ATPase/helicase domain for RNA binding and two caspase activation and recruitment domains (CARDs) located in tandem at the N-terminal end of the protein (4). The CARDs, by way of homotypic interactions, are required for the signaling function between RIG-I and the newly discovered interferon-␤ promoter stimulator-1 (IPS-1), also called Cardif, MAVS, and VISA (for review see Ref. 5). The latter serves as an intermediate to the cascade leading to the activation of NF-B and IRF-3/7, which results in the production of antiviral cytokines such as type-1 interferons (6 -9). The receptor function of RIG-I is non-redundant and therefore essential to coordinate the signaling cascade leading to antiviral response (4), as confirmed by knock-out studies (10). Notably, the Huh7.5 cell line of hepatocytes is particularly permissive to HCV and vesicular stomatitis virus infection as the result of a defective RIG-I protein bearing a single mutation in its first CARD (11); the antiviral sig...

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Structural and functional analysis of the MutS C-terminal tetramerization domain

Cited by 32 publications

References 33 publications

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli

Essential Role of the N-terminal Domain in the Regulation of RIG-I ATPase Activity

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