@article{f72b095a8c3a4565bedf634e7b7a52cd,
title = "RADX prevents genome instability by confining replication fork reversal to stalled forks",
abstract = "RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.",
keywords = "BRCA2, DNA damage, DNA repair, DNA replication, electron microscopy, genome instability, telomere",
author = "Archana Krishnamoorthy and Jessica Jackson and Taha Mohamed and Madison Adolph and Alessandro Vindigni and David Cortez",
note = "Funding Information: We thank Dr. Courtney Lovejoy for suggesting and assisting with the telomere dysfunction assays. We thank Runxiang Zhao and Dr. Wenpeng Liu for creating knockout cells for HLTF, SMARCAL1, and ZRANB3. We thank Dr. Titia de Lange for the long-telomere HeLa cells and Dr. Douglas Bishop for the DLD1 wild-type and BRCA2Δ cells. We thank Dr. Maria Jasin for the RAD51 plasmid. This research was supported primarily by grant R01GM116616 from the NIH to D.C. Additional support came from National Cancer Institute (NCI) grants R01CA237263 and R01CA248526 to A.V. and a Breast Cancer Research Foundation grant to D.C. The EMB Core of grant P01CA092584 provided insect cells expressing MBP-RADX and FLAG-SMARCAL1 proteins used in this study. Funding Information: We thank Dr. Courtney Lovejoy for suggesting and assisting with the telomere dysfunction assays. We thank Runxiang Zhao and Dr. Wenpeng Liu for creating knockout cells for HLTF, SMARCAL1, and ZRANB3. We thank Dr. Titia de Lange for the long-telomere HeLa cells and Dr. Douglas Bishop for the DLD1 wild-type and BRCA2? cells. We thank Dr. Maria Jasin for the RAD51 plasmid. This research was supported primarily by grant R01GM116616 from the NIH to D.C. Additional support came from National Cancer Institute (NCI) grants R01CA237263 and R01CA248526 to A.V. and a Breast Cancer Research Foundation grant to D.C. The EMB Core of grant P01CA092584 provided insect cells expressing MBP-RADX and FLAG-SMARCAL1 proteins used in this study. A.K. performed and analyzed most experiments, with some assistance from T.M. M.A. performed strand exchange and D-loop experiments. J.J. performed the EM experiments and was supervised by A.V. A.K. and D.C. conceived of the project and wrote the manuscript with input from all of the authors. D.C. supervised the project. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2021 Elsevier Inc.",
year = "2021",
month = jul,
day = "15",
doi = "10.1016/j.molcel.2021.05.014",
language = "English",
volume = "81",
pages = "3007--3017.e5",
journal = "Molecular Cell",
issn = "1097-2765",
number = "14",
}