Human Rev1 polymerase disrupts G-quadruplex DNA.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• G-quadruplex DNA (G4 DNA) structures pose challenges for DNA replication.
• The Y-family DNA polymerase Rev1 is crucial for replicating G4 DNA in eukaryotes.

Purpose of the Study:

• To investigate the mechanism by which human Rev1 (hRev1) interacts with and processes G4 DNA.
• To elucidate how hRev1 facilitates replication fork progression at G4 DNA sites.

Main Methods:

• In vitro biochemical assays to assess G4 DNA disruption and refolding.
• Kinetic analyses of hRev1 nucleotidyl transfer activity on G4 DNA substrates.
• Electrophoretic mobility shift assays to determine binding affinities (Kd,DNA) of hRev1 to G4 and non-G4 DNA.

Main Results:

• hRev1 mechanically unfolds G4 DNA structures and inhibits their refolding.
• Nucleotidyl transfer activity is not required for G4 DNA unfolding by hRev1.
• hRev1 exhibits significantly lower binding affinity for G4 DNA compared to non-G4 DNA.
• hRev1 efficiently inserts deoxycytidine monophosphate (dCMP) opposite the first tetrad-guanine in G4 DNA.

Conclusions:

• hRev1 promotes replication fork progression by either unfolding G4 DNA or preventing its refolding.
• These actions of hRev1 are vital for overcoming replication obstacles at G4 DNA.
• The findings highlight a unique role for hRev1 in genome maintenance and G4 DNA metabolism.