Dna2 processes behind the fork long ssDNA flaps generated by Pif1 and replication-dependent strand displacement

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA2 is a crucial enzyme involved in DNA double-strand break (DSB) repair and Okazaki fragment processing.
• Dna2's absence is lethal, but this lethality can be suppressed by inactivating genes like PIF1, POL32, and RAD9.
• Dna2 plays a role in preventing DNA fork reversal and promoting fork restart during replication.

Purpose of the Study:

• To investigate the lethal DNA structures formed upon Dna2 depletion.
• To elucidate the roles of Pif1 and Rad9 in the DNA damage response (DDR) triggered by Dna2 loss.
• To understand how Dna2 processes single-stranded DNA (ssDNA) flaps at replication forks.

Main Methods:

• Depletion of Dna2 in yeast cells.
• Genetic analysis involving PIF1, RAD9, and POL32 gene inactivation.
• Cell cycle arrest analysis and genotoxicity assessment.
• Electron microscopy to visualize DNA structures.
• Analysis of replication fork speed and its effect on DDR.

Main Results:

• Dna2 depletion leads to the accumulation of lethal DNA structures that activate the DDR.
• PIF1 deletion suppresses the lethality of Dna2 depletion.
• RAD9 deletion alleviates the initial cell cycle arrest but results in delayed genotoxicity.
• Slow replication fork speed attenuates the DDR in Dna2-depleted cells.
• Electron microscopy reveals long ssDNA flaps accumulating behind replication forks in Dna2-ablated cells.

Conclusions:

• Dna2 processes long ssDNA flaps generated by Pif1 and lagging strand synthesis to prevent DDR activation.
• Dna2's function in processing ssDNA flaps is essential for genome stability.
• This Dna2 function may be co-opted during Break-Induced Replication (BIR) for double-strand break (DSB) repair.