scholarly journals Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex

2018 ◽  
Author(s):  
Yoori Kim ◽  
Christopher M Furman ◽  
Carol M Manhart ◽  
Eric Alani ◽  
Ilya J Finkelstein
Keyword(s):  
Mismatch Repair ◽  
Catalytic Activities ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Disordered Regions

scholarly journals Handcuffing intrinsically disordered regions in Mlh1-Pms1 disrupts mismatch repair

2021 ◽  
Author(s):  
Christopher M. Furman ◽  
Ting-Yi Wang ◽  
Qiuye Zhao ◽  
Kumar Yugandhar ◽  
Haiyuan Yu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mismatch Repair ◽  
Cross Linking ◽  
Dna Mismatch ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
On Demand ◽  
Transient Loss ◽  
Disordered Regions

AbstractThe DNA mismatch repair (MMR) factor Mlh1-Pms1 contains long intrinsically disordered regions (IDRs). While essential for MMR, their exact functions remain elusive. We performed cross-linking mass spectrometry to identify the major interactions within the Mlh1-Pms1 heterodimer and used this information to insert FRB and FKBP dimerization domains into the IDRs of Mlh1 and Pms1. Yeast bearing these constructs were grown with rapamycin to induce dimerization. Strains containing FRB and FKBP domains in the Mlh1 IDR displayed complete MMR defects when grown with rapamycin, but removing rapamycin restored MMR functions. Furthermore, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization disrupted Mlh1-Pms1 binding to DNA, inappropriately activated Mlh1-Pms1, and caused MMR defects in vivo. We conclude that dynamic and coordinated rearrangements of the MLH IDRs regulate how the complex clamps DNA to catalyze MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.

scholarly journals Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex

2018 ◽  
Author(s):  
Yoori Kim ◽  
Christopher M. Furman ◽  
Carol M. Manhart ◽  
Eric Alani ◽  
Ilya J. Finkelstein
Keyword(s):  
Mismatch Repair ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Facilitated Diffusion ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Associated Proteins ◽  
Dna Substrate ◽  
Disordered Regions

AbstractIntrinsically disordered regions (IDRs) are present in at least 30% of the eukaryotic proteome and are enriched in chromatin-associated proteins. Using a combination of genetics, biochemistry, and single-molecule biophysics, we characterize how IDRs regulate the functions of the yeast MutLα (Mlh1-Pms1) mismatch repair (MMR) complex. Shortening or scrambling the IDRs in both subunits ablates MMR in vivo. Mlh1-Pms1 complexes with shorter IDRs that disrupt MMR retain wild-type DNA binding affinity but are impaired for diffusion on both naked and nucleosome-coated DNA. Moreover, the IDRs also regulate the ATP hydrolysis and nuclease activities that are encoded in the structured N- and C-terminal domains of the complex. This combination of phenotypes underlies the catastrophic MMR defect seen with the mutant MutLα in vivo. More broadly, this work highlights an unanticipated multi-functional role for IDRs in regulating both facilitated diffusion on chromatin and nucleolytic processing of a DNA substrate.

scholarly journals Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vikas A. Tillu ◽  
James Rae ◽  
Ya Gao ◽  
Nicholas Ariotti ◽  
Matthias Floetenmeyer ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Membrane Lipid ◽  
Membrane Curvature ◽  
Caveolin 1 ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Solution Generation ◽  
Endosomal System ◽  
Disordered Regions

AbstractCaveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

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