Affiliation:
1. Institut de Génétique Humaine Université de Montpellier, CNRS, Equipe labélisée Ligue contre le Cancer Montpellier France
2. Université Paris Cité, CNRS, Institut Jacques Monod Paris France
3. Department of Genetics Stanford University Stanford CA USA
4. Institut Universitaire de France (IUF) Paris France
Abstract
AbstractR‐loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H‐deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA‐coated single‐stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU‐arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.
Funder
Agence Nationale de la Recherche
College of Natural Resources and Sciences, Humboldt State University
Fondation ARC pour la Recherche sur le Cancer
Fondation pour la Recherche Médicale
Institut Universitaire de France
Publisher
Springer Science and Business Media LLC
Subject
General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,Molecular Biology,General Neuroscience