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

TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution.

During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.We thank Drs G. Stewart and F. Esashi for cell lines, Professor T.D. Halazonetis, Dr G.J. Gorbsky and Dr G. Stewart for plasmids and antibodies. We also thank Dr C. Lagerholm (Wolfson Imaging Centre, Oxford) and Dr D. Waithe (CBRG, Oxford) for their help with microscopy and image analysis, and the Mass Spectrometry Laboratory (IBB PAS) for their work on analyses of GFP–TOP2A immunoprecipitation experiments. We also thank Professor I. Hickson for helpful comments on the manuscript. This work was funded by a Worldwide Cancer Research International Fellowship (to W.N.), a WIMM/Medical Research Council Senior Non-Clinical Fellowship (to W.N.), a Polish Ministry of Science and Higher Education fellowship (to J.N.) and Polish National Science Center grant N N303 571539 (to J.N.).This is the final published version. It first appeared at http://www.nature.com/ncomms/2015/150312/ncomms7572/full/ncomms7572.html#abstract

TopBP1 interacts with BLM to maintain genome stability but is dispensable for preventing BLM degradation.

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.293FT cells, E1A antibody, and hr703 virus were gifts from Roger Grand, and DT40 cells and human LCLs were gifts from Julian Sale and Ian Hickson, respectively. We thank Nathan Ellis, Thanos Halazonetis, Frank Hänel, and Minoru Takata for plasmids; Grant Stewart and Yi Wang for antibodies; and Gabriel Balmus, Josep Forment, Abderrahmane Kaidi, Christine Schmidt, and Jon Travers for critical reading of the manuscript. This work was funded by a Worldwide Cancer Research International Fellowship and a WIMM/Medical Research Council Senior Non-Clinical Fellowship (MRCG0902418) to W.N., and by Polish Ministry of Science and Higher Education fellowship and Polish National Science Center grant number N303 571539 to J.N. The Jackson lab is funded by Cancer Research UK (CRUK) program grant C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement number HEALTH-F2-2010-259893 (DDResponse). Core infrastructure funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK.This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.molcel.2015.02.01

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.Work in W.N.’s laboratory is funded by ICR Intramural Grant and Cancer Research UK Programme (A24881). R.A.S. and L.S. were supported by WIMM Senior Non-Clinical Fellowship awarded to W.N. M.M.S. and M.A.B. were supported by the Intramural Research Program of the NIH, National Institute on Aging, United States (Z01-AG000746-08). Work in S.G.’s laboratory is supported by BRFAA Intramural Funds. V.C. was supported by John S. Latsis Public Benefit Foundation and Alexander S. Onassis Public Benefit Foundation. Work in the P.P.’s laboratory is supported by grants from the Agence Nationale pour la Recherche (ANR), the Ligue Contre le Cancer (équipe labellisée), SIRIC Montpellier Cancer (INCa Inserm DGOS 12553), and the MSDAvenir fund

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis

Generation of a novel endogenously StrepII/FLAG tagged system for the identification of the vertebrate FANCJ associated proteins

Fanconi Anaemia (FA) is a rare inherited chromosomal instability disorder characterized by developmental abnormalities, bone marrow failure, increased risk of developing cancer and an increased cellular sensitivity to DNA interstrand crosslinking compounds, including the anticancer drugs, cisplatin, nitrogen mustard and mitomycin C. From a therapeutic point of view, it is important to understand how cells respond to and repair DNA damage caused by crosslinking compounds, used to treat cancer. To date, fifteen different proteins involved in a common pathway, known as the FA pathway have been identified and are known to respond to, and influence repair of interstrand crosslinks (ICLs). So far, the role of these proteins in ICL repair remains elusive. To gain insights into the molecular basis of ICL repair, we chose to study the FA-associated helicase FANCJ, as it is known to bind and metabolize a variety of DNA substrates, implicating it in maintenance of genomic stability. Here, I report a novel genetic-proteomic approach to study FANCJ. This system uses epitope tagged FANCJ expressed from its chromosomal locus, in chicken DT40 cells, kept under the control of its endogenous promoter. Tandem affinity purification is employed to purify and isolate the tagged protein and identify interacting partners of interest. To date, I have generated, characterized and validated epitope tagged FANCJ cell lines. Pilot immunoprecipitations were carried out to establish the efficiency and reproducibility of the immunoprecipitations (IPs). Analysis by mass spectrometry revealed the presence of epitope tagged FANCJ in scaled-up immunoprecipitations. In the future, these cell lines will be used to identify and characterize the FANCJ interactome.This thesis is not currently available via ORA