Defining the functional footprint for recognition and repair of deaminated DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Spontaneous DNA deamination is mutagenic if not repaired by the base excision repair (BER) pathway.
• Existing crystallographic data of BER enzymes show compact DNA binding sites but lack information on dynamic interactions and enzyme handoffs.
• Understanding enzyme binding and coordination is crucial for DNA repair mechanisms.

Purpose of the Study:

• To define the DNA binding sites of alkyladenine DNA glycosylase (AAG) and AP endonuclease (APE1), the first two enzymes in the human BER pathway for deaminated purine repair.
• To investigate the functional interactions and coordination between AAG and APE1 during DNA repair.
• To compare functional binding sites with structures obtained from crystallography.

Main Methods:

• Functional footprinting approach was employed to determine the binding sites of AAG and APE1 on DNA.
• Enzyme turnover assays were conducted to assess the interaction and exchange between AAG and APE1.
• Comparison of functional footprint data with existing crystallographic structures.

Main Results:

• The functional footprint of full-length AAG aligns with crystal structures of truncated AAG.
• Full-length APE1 exhibits a significantly larger binding site than observed in crystal structures.
• AAG turnover is enhanced by APE1, indicating rapid enzyme exchange at the repair intermediate.
• Enzyme coordination does not necessitate an extended binding footprint, suggesting independent site engagement.

Conclusions:

• Functional footprinting offers unique insights into enzyme-DNA interactions beyond traditional methods.
• APE1 possesses a larger DNA binding site than previously characterized by crystallography.
• AAG and APE1 rapidly and independently engage the DNA repair site, facilitating efficient base excision repair.