Rebekka A. Schwab

ATR activation and replication fork restart are defective in FANCM‐deficient cells

Fanconi anaemia is a chromosomal instability disorder associated with cancer predisposition and bone marrow failure. Among the 13 identified FA gene products only one, the DNA translocase FANCM, has homologues in lower organisms, suggesting a conserved function in DNA metabolism. However, a precise role for FANCM in DNA repair remains elusive. Here, we show a novel function for FANCM that is distinct from its role in the FA pathway: promoting replication fork restart and simultaneously limiting the accumulation of RPA‐ssDNA. We show that in DT40 cells this process is controlled by ATR and PLK1, and that in the absence of FANCM, stalled replication forks are unable to resume DNA synthesis and genome duplication is ensured by excess origin firing. Unexpectedly, we also uncover an early role for FANCM in ATR‐mediated checkpoint signalling by promoting chromatin retention of TopBP1. Failure to retain TopBP1 on chromatin impacts on the ability of ATR to phosphorylate downstream molecular targets, including Chk1 and SMC1. Our data therefore indicate a fundamental role for FANCM in the maintenance of genome integrity during S phase.

The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription.

Summary DNA replication stress can cause chromosomal instability and tumor progression. One key pathway that counteracts replication stress and promotes faithful DNA replication consists of the Fanconi anemia (FA) proteins. However, how these proteins limit replication stress remains largely elusive. Here we show that conflicts between replication and transcription activate the FA pathway. Inhibition of transcription or enzymatic degradation of transcription-associated R-loops (DNA:RNA hybrids) suppresses replication fork arrest and DNA damage occurring in the absence of a functional FA pathway. Furthermore, we show that simple aldehydes, known to cause leukemia in FA-deficient mice, induce DNA:RNA hybrids in FA-depleted cells. Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity. Failure to activate a response to physiologically occurring DNA:RNA hybrids may critically contribute to the heightened cancer predisposition and bone marrow failure of individuals with mutated FA proteins.

FANCJ couples replication past natural fork barriers with maintenance of chromatin structure

The FANCJ helicase promotes DNA replication in trans by counteracting fork stalling at replication barriers and suppresses heterochromatin spreading by coupling fork movement with maintenance of chromatin structure.

The DNA translocase activity of FANCM protects stalled replication forks

FANCM is the most highly conserved protein within the Fanconi anaemia (FA) tumour suppressor pathway. However, although FANCM contains a helicase domain with translocase activity, this is not required for its role in activating the FA pathway. Instead, we show here that FANCM translocaseactivity is essential for promoting replication fork stability. We demonstrate that cells expressing translocase-defective FANCM show altered global replication dynamics due to increased accumulation of stalled forks that subsequently degenerate into DNA double-strand breaks, leading to ATM activation, CTBP-interacting protein (CTIP)-dependent end resection and homologous recombination repair. Accordingly, abrogation of ATM or CTIP function in FANCM-deficient cells results in decreased cell survival. We also found that FANCM translocase activity protects cells from accumulating 53BP1-OPT domains, which mark lesions resulting from problems arising during replication. Taken together, these data show that FANCM plays an essential role in maintaining chromosomal integrity by promoting the recovery of stalled replication forks and hence preventing tumourigenesis.

The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation.

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3‐K‐generated phosphoinositides, in Theileria‐transformed leucocytes. Akt/PKB is constitutively activated in a PI3‐K‐ and parasite‐dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF‐κB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3‐K‐Akt/PKB pathway and NF‐κB activation, resulting in protection against apoptosis. In Theileria‐transformed leucocytes, activation of the NF‐κB and the PI3‐K‐Akt/PKB pathways are not directly linked. The PI3‐K‐Akt/PKB pathway does not contribute to the persistent induction of IκBα phosphorylation, NF‐κB DNA‐binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF‐κB‐dependent protection against apoptosis is not dependent on a functional PI3‐K‐Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria‐transformed T cells.

Visualization of DNA replication in the vertebrate model system DT40 using the DNA fiber technique.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

TopBP1 Interacts with BLM to Maintain Genome Stability but Is Dispensable for Preventing BLM Degradation

Summary The Bloom syndrome helicase BLM and topoisomerase-IIβ-binding protein 1 (TopBP1) are key regulators of genome stability. It was recently proposed that BLM phosphorylation on Ser338 mediates its interaction with TopBP1, to protect BLM from ubiquitylation and degradation (Wang et al., 2013). Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304. Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability. However, BLM-TopBP1 binding is important for maintaining genome integrity, because in its absence cells display increased sister chromatid exchanges, replication origin firing and chromosomal aberrations. Therefore, the BLM-TopBP1 interaction maintains genome stability not by controlling BLM protein levels, but via another as-yet undetermined mechanism. Finally, we identify critical residues that mediate interactions between TopBP1 and MDC1, and between BLM and TOP3A/RMI1/RMI2. Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network.

BRCA2 coordinates the activities of cell-cycle kinases to promote genome stability.

Summary Numerous human genome instability syndromes, including cancer, are closely associated with events arising from malfunction of the essential recombinase Rad51. However, little is known about how Rad51 is dynamically regulated in human cells. Here, we show that the breast cancer susceptibility protein BRCA2, a key Rad51 binding partner, coordinates the activity of the central cell-cycle drivers CDKs and Plk1 to promote Rad51-mediated genome stability control. The soluble nuclear fraction of BRCA2 binds Plk1 directly in a cell-cycle- and CDK-dependent manner and acts as a molecular platform to facilitate Plk1-mediated Rad51 phosphorylation. This phosphorylation is important for enhancing the association of Rad51 with stressed replication forks, which in turn protects the genomic integrity of proliferating human cells. This study reveals an elaborate but highly organized molecular interplay between Rad51 regulators and has significant implications for understanding tumorigenesis and therapeutic resistance in patients with BRCA2 deficiency.

The IKK Inhibitor BMS-345541 Affects Multiple Mitotic Cell Cycle Transitions

The IκB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFκB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKα also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541, a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell-cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell-cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS 345541 function could be useful for cell-cycle control studies and may provide valuable clues for the design of future therapeutics.

The DNA fibre technique – tracking helicases at work

Faithful duplication of genetic material during every cell division is essential to ensure accurate transmission of genetic information to daughter cells. DNA helicases play a crucial role in promoting this process by facilitating almost all transactions occurring on DNA, including DNA replication and repair. They are responsible not only for DNA double helix unwinding ahead of progressing replication forks but also for resolution of secondary structures like G4 quadruplexes, HJ branch migration, double HJ dissolution, protein displacement, strand annealing and many more. Their importance in maintaining genome stability is underscored by the fact that many human disorders, including cancer, are associated with mutations in helicase genes. Here we outline how DNA fibre fluorography, a straightforward and inexpensive approach, can be employed to study the in vivo function of helicases in DNA replication and the maintenance of genome stability at a single molecule level. This approach directly visualizes the progression of individual replication forks within living cells and hence provides quantitative information on various aspects of DNA synthesis, such as replication fork processivity (replication speed), fork stalling, origin usage and fork termination.