Visualization of recombination-mediated damage bypass by template switching

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA damage poses a threat to genome stability.
• Template switching (TS) is a key mechanism for bypassing DNA lesions during replication.
• PCNA polyubiquitination and polymerase δ are involved in TS.

Purpose of the Study:

• To characterize the intermediates of template switching (TS) in Saccharomyces cerevisiae.
• To elucidate the DNA transitions involved in recombination-mediated DNA damage tolerance.

Main Methods:

• Two-dimensional gel electrophoresis
• Electron microscopy (EM)
• Analysis of a defined chromosome locus in yeast

Main Results:

• Identified five major families of TS intermediates.
• Demonstrated that single-stranded DNA gaps (150-200 nt) initiate TS via strand invasion.
• Observed double Holliday junction-like structures mediating late TS stages before processing to hemicatenanes.

Conclusions:

• TS initiates from single-stranded DNA gaps, not DNA ends.
• Recombination-mediated DNA damage tolerance involves specific DNA transitions.
• Double Holliday junctions play a role in mitotic DNA repair and replication under stress.