Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand

Area of Science:

• Molecular Biology
• DNA Replication
• Biochemistry

Background:

• DNA damage can stall replication forks, hindering DNA replication.
• DNA damage bypass pathways, like template switching, are crucial for cell survival.
• Rad5 helicase in yeast mediates fork reversal, a key step in template switching, but its mechanism is unclear.

Purpose of the Study:

• To elucidate the mechanism of Rad5 helicase in fork reversal.
• To determine Rad5's substrate specificity for different DNA fork structures.
• To understand how Rad5 facilitates template switching during DNA damage bypass.

Main Methods:

• Utilized a Förster Resonance Energy Transfer (FRET)-based assay to monitor fork reversal in real time.
• Investigated Rad5 binding and catalytic activity on various fork DNA substrates.
• Assessed the influence of leading/lagging strand gaps, RPA, and RNA primers on Rad5 function.

Main Results:

• Rad5 helicase preferentially binds and reverses fork DNA substrates with short gaps (10-30 nt) in the leading strand.
• This preference leads to the formation of chicken foot intermediates with 5' overhangs.
• Rad5 activity is modulated by the presence of RPA and RNA primers in the lagging strand.

Conclusions:

• Rad5 exhibits substrate specificity, favoring leading-strand gaps for efficient fork reversal.
• The observed preference facilitates the formation of suitable intermediates for subsequent DNA polymerase extension.
• This study provides mechanistic insights into Rad5 function in DNA damage bypass and template switching.