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

Uncontrolled degradation and collapse of stalled replication forks (RFs) are primary sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse remain to be fully elaborated. Here, we show that polynucleotide kinase-phosphatase (PNKP) localizes at stalled forks and protects stalled forks from excessive degradation. The loss of PNKP results in nucleolytic degradation of nascent DNA at stalled RFs. This mechanism is different from the BRCA2-dependent fork protection pathway, which protects stalled forks from excessive MRE11-dependent nucleolytic degradation. Our research shows that hydroxyurea treatment leads to increased misincorporation of ribonucleotides in DNA, which in turn traps TOP1 on stalled RFs. We have also found that reducing the levels of TOP1 or TDP1 in cells can reverse the degradation of nascent DNA observed in PNKP-deficient cells. In summary, our data suggest that PNKP plays a role in maintaining the stability of stalled RFs.