Abstract SY34-03: Recombination functions of BRCA1 and BRCA2 at stalled replication forks

Abstract

Abstract Replication fork stalling is a common event during scheduled DNA synthesis. Cancer cells, including very early neoplastic lesions, often reveal high levels of replication-related DNA damage (“replication stress”), suggesting that defective processing of stalled forks is an early and near-ubiquitous process in cancer. A number of distinct protein complexes operate at the stalled fork to ensure its error-free processing and to minimize the risk of genomic instability. If the stalled fork response is dysregulated, this can lead to chromosome breakage, misrepair and chromosome rearrangement at the site of replication arrest. Thus, understanding the mechanisms that operate on the stalled fork in health and disease is critical to our understanding of genomic instability in cancer. The proteins that act at the stalled fork are also promising targets for cancer therapy, as suggested by the current testing of ATR and CHK1 inhibitors in clinical trials and by the use of poly(ADP-ribose) polymerase (PARP) inhibitors for therapy of homologous recombination (HR)-defective cancers. The HR pathway plays an essential role in preventing genomic instability at stalled forks. HR proteins both stabilize the stalled fork and mediate error-free repair of broken forks. Consistent with this, germ line mutation of HR genes can cause hereditary developmental disorders and hereditary cancer predisposition syndromes. A large number of these genes are now recognized, some of the most prominent being the hereditary breast/ovarian cancer predisposition genes BRCA1 and BRCA2, the Fanconi anemia (FA) genes and the Bloom's syndrome gene (BLM). Much has been learned about HR from analysis of the repair of “generic” chromosomal double strand breaks (DSBs)—for example, those induced by the rare-cutting homing endonuclease I-SceI. However, until recently, there were no tractable tools for analyzing HR at a molecular level at stalled replication forks on a mammalian chromosome. We solved this longstanding problem by adapting the Escherichia coli Tus/Ter complex to induce site-specific replication fork stalling and chromosomal HR in mammalian cells, making this process accessible to quantitative molecular analysis (Willis et al. 2014). We found that the mechanisms that control HR at stalled forks differ from those operating on a generic DSB, especially with regard to aberrant HR responses. Specifically, in BRCA/HR-defective cells, the majority of HR products at Tus/Ter-stalled forks resolve via an error-prone HR pathway termed “long tract” gene conversion (LTGC). Surprisingly, Tus/Ter-induced LTGC frequencies are paradoxically increased in BRCA mutants in comparison to wild type cells—an effect that is not seen in response to a generic DSB. Long tract gene conversion has similarity to “break-induced replication” (BIR) in yeast and is a potential mediator of genomic instability. Thus, the stalled fork is uniquely vulnerable to BIR-type mechanisms of genomic instability. Our recent work is focused on a novel aberrant outcome of replication arrest: the non-homologous tandem duplication (TD). Nonhomologous TDs are frequent in some cancer genomes and may be significant mediators of gene dysregulation in cancer. We have identified BRCA1, acting in parallel with two replication fork helicases, FANCM and BLM, as a critical suppressor of nonhomologous TD formation at Tus/Ter-stalled forks. The Tus/Ter system may provide a useful model of TD formation in cancer. In this presentation, we will address progress in identifying the gene network that normally suppresses TDs at stalled replication forks. We will also discuss our efforts to determine the underlying mechanisms, including evidence of chromosome breakage at sites of replication arrest. Willis NA, Chandramouly G, Huang B, Kwok A, Follonier C, Deng C, Scully R. 2014. BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. Nature 510: 556-559. Citation Format: Ralph Scully, Nicholas A. Willis. Recombination functions of BRCA1 and BRCA2 at stalled replication forks. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY34-03.