AND-1 fork protection function prevents fork resection and is essential for proliferation

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• AND-1/Ctf4 acts as a crucial link between the CMG helicase and DNA polymerase alpha, essential for DNA replication.
• Replication stress can lead to DNA damage and genomic instability.

Purpose of the Study:

• To investigate the role of AND-1/Ctf4 in cell proliferation and genome stability.
• To elucidate the specific functions of AND-1/Ctf4 domains during DNA replication.

Main Methods:

• Utilized an inducible degron system in avian cells to deplete AND-1.
• Analyzed cell cycle progression, DNA damage, and replication fork dynamics.
• Performed domain analysis of AND-1 to identify functional regions.

Main Results:

• AND-1 depletion caused G2 cell cycle arrest with activated DNA damage checkpoints.
• Replication without AND-1 led to slowed replication fork speed and accumulation of single-stranded DNA (ssDNA) gaps.
• These ssDNA gaps were converted to double-strand breaks (DSBs) in G2.
• Inhibition of MRE11 nuclease with mirin reverted fork resection and DNA damage accumulation, but not fork slowdown.
• The WD40 domain of AND-1 was essential for preventing fork resection and subsequent DSB-associated lethality, while the HMG box was important for replication speed.

Conclusions:

• AND-1/Ctf4 possesses a critical fork protection function mediated by its WD40 domain, essential for cell proliferation.
• This function averts genome instability by preventing excessive DNA resection and DSB formation.
• AND-1/Ctf4 plays a dual role in replication: facilitating processivity and protecting against fork collapse.