Sequential and multistep substrate interrogation provides the scaffold for specificity in human flap endonuclease 1

Area of Science:

• Molecular Biology
• Biochemistry
• Enzymology

Background:

• Human flap endonuclease 1 (FEN1) is a key enzyme in DNA replication, repair, and recombination.
• FEN1 belongs to the structure-specific 5' nucleases, which share common features but act on diverse DNA substrates.
• These enzymes cleave DNA at specific positions relative to 5' end junctions.

Purpose of the Study:

• To elucidate the mechanism by which FEN1 recognizes and processes DNA substrates.
• To understand how FEN1's action contributes to the unifying mechanisms of 5' nucleases.
• To investigate how FEN1 ensures specificity and protects DNA from unintended cleavage.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) was employed to observe FEN1-DNA interactions at the single-molecule level.
• The study tracked the dynamic steps of substrate recognition and DNA bending induced by FEN1.
• Analysis focused on the coordination of substrate features, threading into the gateway, and binding at the junction base.

Main Results:

• FEN1 utilizes a multistep mechanism to verify all features of a DNA substrate before inducing DNA bending.
• The enzyme coordinates threading of the 5' flap into a conserved gateway and binds at the junction base to facilitate bending.
• This sequential recognition ensures that DNA bending is restricted to a common final step across different 5' nucleases.

Conclusions:

• The findings reveal a sequential substrate recognition process in FEN1, crucial for its function in DNA metabolism.
• This mechanism allows for diverse substrate recognition by different 5' nucleases while maintaining a conserved DNA bending step.
• The study highlights how FEN1's precise mechanism protects DNA junctions from aberrant bending and cleavage, ensuring genome integrity.