53BP1 Mediates ATR-Chk1 Signaling and Protects Replication Forks under Conditions of Replication Stress

Abstract

Complete replication of the genome is an essential prerequisite for normal cell division, but a variety of factors can block the replisome, triggering replica-tion stress and potentially causing mutation or cell death. The cellular response to replication stress involves recruitment of proteins to stabilize the replication fork and transmit a stress signal to pause the cell cycle and allow fork restart. We find that the ubiquitously expressed DNA damage response factor 53BP1 is required for the normal response to replication stress. Using primary, ex vivo B cells, we showed that a population of 53BP1 / cells in early S phase is hypersensitive to short-term exposure to three different agents that induce replication stress. 53BP1 localizes to a subset of replication forks following induced replication stress, and an absence of 53BP1 leads to defective ATR-Chk1-p53 signaling and caspase 3-mediated cell death. Nas-cent replicated DNA additionally undergoes degradation in 53BP1 / cells. These results show that 53BP1 plays an important role in protecting replication forks during the cellular response to replication stress, in addition to the previously characterized role of 53BP1 in DNA double-strand break repair. M aintenance of genomic integrity relies on effective cellular responses to DNA damage. Although DNA damage can be a consequence of exogenous mutagens, such as ionizing radiation, a significant amount of damage also arises as a result of endogenous cellular processes, such as normal oxidative metabolism. One key cellular process that can contribute to DNA damage is DNA replication (1). During a typical S phase, cells encounter challenges to replication, such as extended repetitive genomic regions or exogenous replication poisons, which can cause replication forks to stall. Such obstacles to normal replication represent replication stress (2). Replication stress triggers an S-phase cell cycle checkpoint, during which late replication origins do not fire, and specific cellular responses stabilize the fork and prevent collapse and formation of DNA double-strand breaks (DSBs). During the cellular replication stress response, a number of changes take place at the replication fork. The minichromosome maintenance (MCM) helicase uncouples from the replicative DNA polymerase, and a region of single-stranded DNA is formed, coated with replication protein A (RPA) (3). The fork protection complex, a heterodimer of Timeless and Tipin, binds to RPA at single-stranded DNA, helping to stabilize the fork and prevent dissociation of the replisome components (4). PCNA, which normally ensures processivity of the replication fork, becomes ubiquitylated and is displaced (5, 6). A large number of other proteins, including damage signaling molecules and repair factors, become enriched at stalled forks (7). In some cases, replication forks reverse, by unwinding of newly synthesized DNA from its homologous template followed by annealing to form a fourth regressed "arm" (8, 9). Loading of RAD51 at reversed forks