Handcuffing intrinsically disordered regions in Mlh1-Pms1 disrupts mismatch repair

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The DNA mismatch repair (MMR) pathway is essential for genomic stability.
• Mlh1-Pms1 is a key heterodimer in the MMR machinery.
• Intrinsically disordered regions (IDRs) within Mlh1-Pms1 have poorly understood roles.

Purpose of the Study:

• To investigate the in vivo function of Mlh1-Pms1 IDRs in DNA mismatch repair.
• To elucidate the role of IDR dynamics in regulating MMR complex activity.
• To apply inducible dimerization as a tool to probe IDR function.

Main Methods:

• Cross-linking mass spectrometry to identify protein interactions.
• Engineered yeast strains expressing Mlh1-Pms1 with inserted FRB and FKBP dimerization domains within IDRs.
• Inducible dimerization using rapamycin to control protein interactions and assess MMR function.

Main Results:

• Rapamycin-induced dimerization of FRB/FKBP domains within the Mlh1 IDR caused a complete MMR defect.
• Dimerization between Mlh1 and Pms1 IDRs led to rapamycin-dependent defects in endonuclease activity.
• Linking Mlh1 and Pms1 IDRs inappropriately activated endonuclease activity.

Conclusions:

• Dynamic and coordinated rearrangements of MLH IDRs positively and negatively regulate MMR.
• The FRB-FKBP system effectively probes in vivo functions of IDRs in DNA repair complexes.
• This approach enables the study of transient loss of MMR on demand.