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

Fatemeh Mashayekhi; Cassandra Ganje; Marie-Christine Caron; Joshua R Heyza; Yuandi Gao; Elham Zeinali; Mesfin Fanta; Lei Li; Jana Ali; Sofiane Yacine Mersaoui; Jens C Schmidt; Roseline Godbout; Jean-Yves Masson; Michael Weinfeld; Ismail Hassan Ismail

doi:10.1016/j.celrep.2024.115066

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

Cell Rep. 2024 Dec 12;43(12):115066. doi: 10.1016/j.celrep.2024.115066. Online ahead of print.

Authors

Affiliations

¹ Department of Oncology, Faculty of Medicine & Dentistry, University of Alberta, 11560 University Avenue, Edmonton, AB T6G 1Z2, Canada.
² Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University, 9 McMahon, Québec City, QC G1R 3S3, Canada.
³ Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, MI, USA.
⁴ Department of Oncology, Faculty of Medicine & Dentistry, University of Alberta, 11560 University Avenue, Edmonton, AB T6G 1Z2, Canada; Biophysics Department, Faculty of Science, Cairo University, Giza 12613, Egypt. Electronic address: iismail@ualberta.ca.

PMID: 39671289
DOI: 10.1016/j.celrep.2024.115066

Abstract

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.

Keywords: CP; DNA repair; EXO1; Fork reversal; MRE11; Molecular biology; PNKP; Topoisomerase 1; hydroxyurea; replication stress; ribonucleotide misincorporation.