TopBP1/Rad4/Dpb11 is an essential eukaryotic protein with important roles in DNA replication, DNA repair, DNA damage checkpoint activation, and chromosome segregation. TopBP1 serves as a scaffold to ...assemble protein complexes in a phosphorylation-dependent manner via its multiple BRCT-repeats. Recently, it has become clear that TopBP1 is repurposed to scaffold different processes dependent on cell cycle regulated changes in phosphorylation of client proteins. Here we review the functions of human TopBP1 in maintaining genome integrity during mitosis.
Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The ...critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.
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•A native Fanconi anaemia core complex is purified from vertebrate cells•Purified FA core complex monoubiquitinates FANCD2 in vitro•FANCB, FANCL, and FAAP100 constitute the minimal monoubiquitination module•Other subunits contribute to maximal activity and site specificity of FANCL
FANCD2 monoubiquitination by the multisubunit Fanconi anaemia (FA) core complex is the key activating event in a conserved DNA repair pathway. Rajendra et al. now purify the core complex and reconstitute the monoubiquitination event in vitro. Genetic and biochemical dissection define a three-component subcomplex that functions as the catalytic monoubiquitination module.
Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome ...segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis.
CIP2A is an inhibitor of the tumour suppressor protein phosphatase 2A. Recently, CIP2A was identified as a synthetic lethal interactor of BRCA1 and BRCA2 and a driver of basal-like breast cancers. In ...addition, a joint role of TopBP1 (topoisomerase IIβ-binding protein 1) and CIP2A for maintaining genome integrity during mitosis was discovered. TopBP1 has multiple functions as it is a scaffold for proteins involved in DNA replication, transcriptional regulation, cell cycle regulation and DNA repair. Here, we briefly review details of the CIP2A-TopBP1 interaction, its role in maintaining genome integrity, its involvement in cancer and its potential as a therapeutic target.
Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an ...unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase-mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1's role in HR, with potential clinical implications for cancer treatment.
Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of ...toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.
Proteostasis can be disturbed by mutations affecting folding and stability of the encoded protein. An example is the ubiquitin ligase Parkin, where gene variants result in autosomal recessive ...Parkinsonism. To uncover the pathological mechanism and provide comprehensive genotype-phenotype information, variant abundance by massively parallel sequencing (VAMP-seq) is leveraged to quantify the abundance of Parkin variants in cultured human cells. The resulting mutational map, covering 9219 out of the 9300 possible single-site amino acid substitutions and nonsense Parkin variants, shows that most low abundance variants are proteasome targets and are located within the structured domains of the protein. Half of the known disease-linked variants are found at low abundance. Systematic mapping of degradation signals (degrons) reveals an exposed degron region proximal to the so-called "activation element". This work provides examples of how missense variants may cause degradation either via destabilization of the native protein, or by introducing local signals for degradation.
Transcription-replication (T-R) conflicts are profound threats to genome integrity. However, whilst much is known about the existence of T-R conflicts, our understanding of the genetic and temporal ...nature of how cells respond to them is poorly established. Here, we address this by characterizing the early cellular response to transient T-R conflicts (TRe). This response specifically requires the DNA recombination repair proteins BLM and BRCA2 as well as a non-canonical monoubiquitylation-independent function of FANCD2. A hallmark of the TRe response is the rapid co-localization of these three DNA repair factors at sites of T-R collisions. We find that the TRe response relies on basal activity of the ATR kinase, yet it does not lead to hyperactivation of this key checkpoint protein. Furthermore, specific abrogation of the TRe response leads to DNA damage in mitosis, and promotes chromosome instability and cell death. Collectively our findings identify a new role for these well-established tumor suppressor proteins at an early stage of the cellular response to conflicts between DNA transcription and replication.
Collision between the molecular machineries responsible for transcription and replication is an important source of genome instability. Certain transcribed regions known as chromosomal fragile sites ...are particularly prone to recombine and mutate in a manner that correlates with specific transcription and replication patterns. At the same time, these chromosomal fragile sites engage in aberrant DNA structures in mitosis. Here, we discuss the mechanistic details of transcription–replication conflicts including putative scenarios for R-loop-induced replication inhibition to understand how transcription–replication conflicts transition from S phase into various aberrant DNA structures in mitosis.
DNA anaphase bridges are a potential source of genome instability that may lead to chromosome breakage or nondisjunction during mitosis. Two classes of anaphase bridges can be distinguished: ...DAPI-positive chromatin bridges and DAPI-negative ultrafine DNA bridges (UFBs). Here, we establish budding yeast Saccharomyces cerevisiae and the avian DT40 cell line as model systems for studying DNA anaphase bridges and show that TopBP1/Dpb11 plays an evolutionarily conserved role in their metabolism. Together with the single-stranded DNA binding protein RPA, TopBP1/Dpb11 binds to UFBs, and depletion of TopBP1/Dpb11 led to an accumulation of chromatin bridges. Importantly, the NoCut checkpoint that delays progression from anaphase to abscission in yeast was activated by both UFBs and chromatin bridges independently of Dpb11, and disruption of the NoCut checkpoint in Dpb11-depleted cells led to genome instability. In conclusion, we propose that TopBP1/Dpb11 prevents accumulation of anaphase bridges via stimulation of the Mec1/ATR kinase and suppression of homologous recombination.
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