Flap endonuclease 1 mechanism analysis indicates flap base binding prior to threading

Area of Science:

• Molecular Biology
• Enzymology
• DNA Repair Mechanisms

Background:

• Flap Endonuclease 1 (FEN1) is crucial for DNA replication and repair.
• FEN1 processes 5' flaps, creating nicks essential for DNA ligation.
• Previous models suggested FEN1 enters flaps from the 5'-end and tracks to the base.

Purpose of the Study:

• To investigate the binding and cleavage mechanism of FEN1 on flap substrates.
• To determine the role of flap length and 5'-end accessibility in FEN1 interaction.
• To challenge and refine existing models of FEN1 substrate processing.

Main Methods:

• Biochemical assays measuring FEN1 binding affinity to various flap substrates.
• Experiments using blocked 5'-ends (biotin/streptavidin) to assess binding mechanisms.
• Sequestration assays with competing substrates to evaluate FEN1-substrate interactions.

Main Results:

• FEN1 exhibits high affinity for the base of long flaps, irrespective of 5'-end blocking.
• FEN1 bound to blocked flaps is more susceptible to sequestration, suggesting a base-first interaction.
• Short flaps (2-4 nucleotides) are cleaved without a significant threading requirement, unlike longer flaps.

Conclusions:

• FEN1 likely binds flap substrates at the base first, then threads the flap through the protein.
• The threading mechanism is dependent on flap length, occurring for flaps >4 nucleotides.
• Short flaps are processed differently, potentially bypassing the threading step, revising current understanding of FEN1 function.