PNKP safeguards stalled replication forks from nuclease-dependent degradation during replication stress

Area of Science:

• Molecular Biology
• Genetics
• DNA Repair

Background:

• Genomic instability arises from uncontrolled degradation and collapse of stalled replication forks (RFs).
• Mechanisms protecting RFs from degradation and collapse are not fully understood.
• Existing pathways like BRCA2-dependent fork protection involve MRE11-dependent nucleolytic degradation.

Purpose of the Study:

• To elucidate the molecular mechanisms of RF protection.
• To investigate the role of polynucleotide kinase-phosphatase (PNKP) in maintaining RF stability.
• To understand how ribonucleotide misincorporation affects RF stability.

Main Methods:

• Localization studies of PNKP at stalled RFs.
• Analysis of nascent DNA degradation in PNKP-deficient cells.
• Investigating the impact of hydroxyurea treatment on DNA and trapped proteins.
• Assessing the effects of reducing TOP1 or TDP1 levels on DNA degradation.

Main Results:

• PNKP localizes to stalled RFs and protects them from excessive degradation.
• Loss of PNKP causes nucleolytic degradation of nascent DNA at stalled RFs.
• Hydroxyurea treatment increases ribonucleotide misincorporation, trapping TOP1 on stalled RFs.
• Reducing TOP1 or TDP1 levels reverses nascent DNA degradation in PNKP-deficient cells.

Conclusions:

• PNKP plays a crucial role in maintaining the stability of stalled replication forks.
• PNKP-mediated fork protection is distinct from the BRCA2-dependent pathway.
• Ribonucleotide metabolism and associated protein interactions (TOP1, TDP1) are critical for RF stability in the absence of PNKP.