Pathway choice in DNA double strand break repair:Observations of a balancing act

Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment.</p

Pathway choice in DNA double strand break repair:Observations of a balancing act

Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment.</p

Pathway choice in DNA double strand break repair: Observations of a balancing act

Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment

Elevated APOBEC3B expression drives a kataegic-like mutation signature and replication stress-related therapeutic vulnerabilities in p53-defective cells

BACKGROUND: Elevated APOBEC3B expression in tumours correlates with a kataegic pattern of localised hypermutation. We assessed the cellular phenotypes associated with high-level APOBEC3B expression and the influence of p53 status on these phenotypes using an isogenic system. METHODS: We used RNA interference of p53 in cells with inducible APOBEC3B and assessed DNA damage response (DDR) biomarkers. The mutational effects of APOBEC3B were assessed using whole-genome sequencing. In vitro small-molecule inhibitor sensitivity profiling was used to identify candidate therapeutic vulnerabilities. RESULTS: Although APOBEC3B expression increased the incorporation of genomic uracil, invoked DDR biomarkers and caused cell cycle arrest, inactivation of p53 circumvented APOBEC3B-induced cell cycle arrest without reversing the increase in genomic uracil or DDR biomarkers. The continued expression of APOBEC3B in p53-defective cells not only caused a kataegic mutational signature but also caused hypersensitivity to small-molecule DDR inhibitors (ATR, CHEK1, CHEK2, PARP, WEE1 inhibitors) as well as cisplatin/ATR inhibitor and ATR/PARP inhibitor combinations. CONCLUSIONS: Although loss of p53 might allow tumour cells to tolerate elevated APOBEC3B expression, continued expression of this enzyme might impart a number of therapeutic vulnerabilities upon tumour cells.British Journal of Cancer advance online publication, 23 May 2017; doi:10.1038/bjc.2017.133 www.bjcancer.com

HSF2BP negatively regulates homologous recombination in DNA interstrand crosslink repair

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that ectopic production of HSF2BP, a BRCA2-interacting protein required for meiotic HR during mouse spermatogenesis, in non-germline human cells acutely sensitize them to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors, resulting in a phenotype characteristic of cells from Fanconi anemia (FA) patients. We biochemically recapitulate the suppression of ICL repair and establish that excess HSF2BP compromises HR by triggering the removal of BRCA2 from the ICL site and thereby preventing the loading of RAD51. This establishes ectopic expression of a wild-type meiotic protein in the absence of any other protein-coding mutations as a new mechanism that can lead to an FA-like cellular phenotype. Naturally occurring elevated production of HSF2BP in tumors may be a source of cancer-promoting genomic instability and also a targetable vulnerability

Attenuated XPC expression is not associated with impaired DNA repair in bladder cancer

Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors

On the Mechanism of Hyperthermia-Induced BRCA2 Protein Degradation

The DNA damage response (DDR) is a designation for a number of pathways that protects our DNA from various damaging agents. In normal cells, the DDR is extremely important for maintaining genome integrity, but in cancer cells these mechanisms counteract therapy-induced DNA damage. Inhibition of the DDR could therefore be used to increase the efficacy of anti-cancer treatments. Hyperthermia is an example of such a treatment—it inhibits a sub-pathway of the DDR, called homologous recombination (HR). It does so by inducing proteasomal degradation of BRCA2 —one of the key HR factors. Understanding the precise mechanism that mediates this degradation is important for our understanding of how hyperthermia affects therapy and how homologous recombination and BRCA2 itself function. In addition, mechanistic insight into the process of hyperthermia-induced BRCA2 degradation can yield new therapeutic strategies to enhance the effects of local hyperthermia or to inhibit HR. Here, we investigate the mechanisms driving hyperthermia-induced BRCA2 degradation. We find that BRCA2 degradation is evolutionarily conserved, that BRCA2 stability is dependent on HSP90, that ubiquitin might not be involved in directly targeting BRCA2 for protein degradation via the proteasome, and that BRCA2 degradation might be modulated by oxidative stress and radical scavengers

HSF2BP Interacts with a Conserved Domain of BRCA2 and Is Required for Mouse Spermatogenesis

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability, and DNA interstrand crosslink repair in vertebrates. We identify HSF2BP, a protein previously described as testis specific and not characterized functionally, as an interactor of BRCA2 in mouse embryonic stem cells, where the 2 proteins form a constitutive complex. HSF2BP is transcribed in all cultured human cancer cell lines tested and elevated in some tumor samples. Inactivation of the mouse Hsf2bp gene results in male infertility due to a severe HR defect during spermatogenesis. The BRCA2-HSF2BP interaction is highly evolutionarily conserved and maps to armadillo repeats in HSF2BP and a 68-amino acid region between the BRC repeats and the DNA binding domain of human BRCA2 (Gly2270-Thr2337) encoded by exons 12 and 13. This region of BRCA2 does not harbor known cancer-associated missense mutations and may be involved in the reproductive rather than the tumor-suppressing function of BRCA2. BRCA2 is a key homologous recombination mediator in vertebrates. Brandsma et al. show that it directly interacts with a testis-expressed protein, HSF2BP, and that male mice deficient for HSF2BP are infertile due to a meiotic recombination defect. They also find that HSF2BP contributes to DNA repair in mouse embryonic stem cells

Pathway choice in DNA double strand break repair: Observations of a balancing act

textabstractProper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment