Vibe H. Oestergaard

TOPBP1 is a scaffold protein with multiple functions in genome integrity. During mitosis, TOPBP1 is coordinating DNA repair and genome maintenance, thereby reducing carry-over of DNA damage to daughter cells. This function emerges as a critical backup pathway in BRCA-deficient cells, yet many aspects of TOPBP1 regulation during mitosis remain unclear. Mitotic kinases are involved in regulating genome maintenance pathways during mitosis. Indeed, TOPBP1 binds the mitotic kinase PLK1, but the molecular aspects as well as the functional significance of this is unclear. To address this, we here identify and characterize a conserved CDK1-primed binding site for PLK1 in TOPBP1. We show that mutation of this results in increased transmission of DNA damage to G1 daughter cells, deficient mitotic DNA repair synthesis, mislocalization of DNA polymerase θ, and increased frequency of anaphase bridges and micronucleation. Consistent with this, BRCA2 depleted cells are highly dependent on TOPBP1–PLK1 complex formation. Altogether, we reveal the molecular details of TOPBP1–PLK1 complex formation and address its role in genome maintenance during mitosis.

The majority of cancer cells experience replication stress, which ultimately causes them to enter mitosis with underreplicated DNA. To alleviate the consequences of replication stress, cells utilize a mechanism known as MiDAS that functions to complete synthesis of underreplicated DNA in early mitosis. This process is considered an Achilles heel for highly replicative cancers. In this study, we show that human TopBP1 localizes to sites of underreplicated DNA marked by FANCD2 and promotes MiDAS through recruitment of the nuclease scaffold protein SLX4. Additionally, we demonstrate that the recruitment of SLX4 to TopBP1 foci in mitosis depends on TopBP1-K704, SLX4-T1260, and several SUMO-interaction motifs in SLX4. Lastly, we show that the recruitment of SLX4 to TopBP1 foci in mitosis is important to prevent transmission of DNA damage to daughter cells. Based on this, we hypothesize that targeting the TopBP1-SLX4 interaction in mitosis may be a potential strategy for anti-cancer therapy.

Recent research reveals that eukaryotic genomes form circular DNA from all parts of their genome, some large enough to carry whole genes. In organisms like yeast and in human cancers, it is often observed that extrachromosomal circular DNA (eccDNA) benefits the individual cell by providing resources for rapid cellular growth. However, our comprehension of eccDNA remains incomplete, primarily due to their transient nature. Early studies suggest they arise when DNA breaks and is subsequently repaired incorrectly. In this review, we provide an overview of the evidence for molecular mechanisms that lead to eccDNA formation in human cancers and yeast, focusing on nonhomologous end joining, alternative end joining, and homologous recombination repair pathways. Furthermore, we present hypotheses in the form of molecular eccDNA formation models and consider cellular conditions which may affect eccDNA generation. Finally, we discuss the framework for future experimental evidence.

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.

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.

The delicate balance of protein homeostasis can be disturbed by mutations that affect folding and stability of the encoded protein. More than half of disease-causing missense variants are thought to lead to protein degradation, but determining which and the molecular mechanisms involved remain enigmatic. To examine these issues, we selected the ubiquitin-protein ligase Parkin, where known missense variants result in an autosomal recessive, early onset Parkinsonism. We used the variant abundance by massively parallel sequencing (VAMP-seq) approach to quantify the abundance of Parkin missense 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, show that most low abundance variants are located within the structured domains of the protein, while the flexible linker regions are more tolerant. The vast majority of low abundance Parkin variants are degraded through the ubiquitin-proteasome system and are stabilized at a lowered temperature. The cellular abundance data correlate with thermodynamic stability, evolutionary conservation, and show that half of the known disease-linked variants are found at low abundance. Systematic mapping of degradation signals (degrons) shows that inherent primary degrons in Parkin largely overlap with regions that are buried, and highly sensitive to mutations. An exposed degron region proximal to the so-called “activation element” is enhanced by substitutions to hydrophobic residues and destroyed by introduction of hydrophilic residues. The data provide examples of how missense variants may cause degradation either via destabilization of the native protein, or by introducing local signals for degradation. Combined with the computational methods based on Parkin structure and conservation, our abundance map sheds light on the mechanisms that cause loss of function, and points to variants where function potentially can be restored.

TopBP1 is a large scaffold protein with multiple functions in genome integrity. We previously identified a novel role for TopBP1 during M phase by showing that TopBP1 reduces carry-over of DNA damage to daughter cells. This function emerges as a critical backup pathway in BRCA deficient cells, yet many aspects of TopBP1 regulation during mitosis are unclear. The mitotic kinase PLK1 has been reported to interact with TopBP1 but the functional relevance of this is unclear. Here, we identify and characterize a conserved PLK1 docking site in TopBP1. Endogenous deletion of the PLK1 docking site in TopBP1 results in increased number of mitotic TopBP1 foci, increased DNA damage in daughter cells, deficient mitotic DNA repair synthesis and increased frequency of binucleation. At the same time, cell cycle distribution and ATR activation are normal in cells with the PLK1 docking site deletion in TopBP1. Interestingly, mutation of this site in TopBP1 renders cells sensitive to PARP inhibitors but not to camptothecin hinting to different cellular effects of the two chemotherapeutics. Altogether, our data indicate that the PLK1-TopBP1 interaction is critical for the mitotic function of TopBP1.

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.

Common chromosomal fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes during conditions of replication stress. Moreover, CFSs are hotspots for deletions and amplifications in cancer genomes. Fragility at CFSs is caused by transcription of extremely large genes, which contributes to replication problems. These extremely large genes do not encode large proteins, but the extreme sizes of the genes originate from vast introns. Intriguingly, the intron sizes of extremely large genes are conserved between mammals and birds. Here, we have used reverse genetics to address the function and significance of the largest intron in the extremely large gene PRKN, which is highly fragile in our model system. Specifically, we have introduced an 80-kilobase deletion in intron 7 of PRKN. We find that gene expression of PRKN is largely unaffected by this intronic deletion. Strikingly, the intronic deletion, which leads to a 12% reduction of the overall size of the PRKN gene body, results in an almost twofold reduction of the PRKN fragility. Our results stress that while the large intron clearly contributes to the fragility of PRKN, it does not play an important role for PRKN expression. Taken together, our findings further add to the mystery concerning conservation of the seemingly non-functional but troublesome large introns in PRKN.

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 promoting 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 loading to 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.