Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis

Area of Science:

• DNA repair mechanisms
• Oxidative DNA damage
• Human genetics and disease

Background:

• MUTYH DNA glycosylase is crucial for repairing adenine misincorporations opposite 8-oxo-7,8-dihydroguanine (8-oxoG), preventing G>T transversions.
• Defective MUTYH activity is linked to MUTYH-associated polyposis (MAP), a condition increasing colorectal cancer risk.
• Specific MUTYH variants (R171W, E466del, 137insIW, Y165C, G382D) found in MAP patients require functional assessment.

Purpose of the Study:

• To investigate the repair capacity of clinically relevant MUTYH variants.
• To characterize the substrate binding and enzymatic activity of these variants.
• To evaluate the utility of surface plasmon resonance (SPR) for assessing MUTYH function.

Main Methods:

• Expression and purification of wild-type and mutant human MUTYH proteins.
• Standard glycosylase assays to measure adenine removal from 8-oxoG:A substrates.
• Surface plasmon resonance (SPR) to analyze real-time binding kinetics (association/dissociation) of MUTYH variants to the 8-oxoG:A DNA substrate.

Main Results:

• R171W, E466del, and Y165C variants exhibited severely reduced binding affinity to the 8-oxoG:A substrate.
• 137insIW and G382D variants showed a slight decrease in binding, primarily due to slower association rates.
• Reduced binding correlated with impaired glycosylase activity; adenine removal was abrogated in R171W, E466del, Y165C, and partially reduced in 137insIW, G382D.

Conclusions:

• The study demonstrates that SPR is a sensitive method for detecting altered substrate recognition and enzymatic activity in MUTYH variants.
• R171W, E466del, and Y165C variants possess significantly compromised DNA repair capacity.
• These findings provide insights into the molecular basis of MAP and highlight the importance of functional assessment for genetic variants.