Human Exonuclease 1 Threads 5'-Flap Substrates through Its Helical Arch

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Human exonuclease 1 (hEXO1) is a 5 e2 -nuclease superfamily member crucial for DNA repair.
• hEXO1 functions as both a 5 e2 -exonuclease and an endonuclease.
• Specificity of hEXO1 and hFEN1 for discontinuous DNA substrates like 5 e2 -flaps has been debated.

Purpose of the Study:

• To investigate the mechanism by which hEXO1 recognizes and processes discontinuous DNA substrates.
• To determine if hEXO1 utilizes a threading mechanism similar to hFEN1.

Main Methods:

• Functional assays using modified DNA substrates with bulky adducts (streptavidin) to probe the hEXO1 active site.
• Kinetic analysis of hEXO1 activity on flap DNA substrates.
• Comparison of hEXO1 and hFEN1 mechanisms.

Main Results:

• hEXO1 threads 5 e2 -flaps through its helical arch, excluding continuous single strands.
• Blocking the arch with streptavidin significantly slowed hEXO1 activity.
• A preformed hEXO1-flap DNA complex showed both fast and slow decay fractions, indicating threaded and unthreaded states.

Conclusions:

• hEXO1 employs a threading mechanism via its helical arch for processing 5 e2 -flap DNA substrates.
• This threading mechanism is conserved among 5 e2 -nuclease superfamily members acting on discontinuous DNA.
• Intrinsic disorder in the arch region may facilitate flap threading without requiring external energy sources.