Biology subjects

Kanaar, R.

Publications and source records attributed to Kanaar, R..

15 recordsLinked to original sources

bioRxiv2026

BRCA2 and RAD54B FxPP motifs Bind DMC1 Filaments through Persistent and Transient Interfaces

During meiosis, double-strand DNA breaks are programmed to initiate homologous recombination. The DMC1 recombinogenic filament is central to the repair of these breaks. However, the three-dimensional structures of the protein-protein interfaces regulating the assembly and activity of this filament have not been described yet. We and others previously reported that in BRCA2, a P-motif called PhePP binds to DMC1 in its oligomeric and filament states. Here we identified a similar P-motif in the DNA translocase RAD54B. We solved the cryo-electron microscopy (cryo-EM) structures of BRCA2 and RAD54B P-motif peptides bound to a ssDNA-DMC1 filament at 1.9-2.0 [A] resolution. While these peptides only share the sequence F-[IV]-P-P, they bind to the filament through larger 9-10 residue core sequences with superimposable structures. Both peptides bind to a hydrophobic and negatively charged site, named the P-site, on a single DMC1 protomer and stabilize the ssDNA-DMC1 filament. Mutagenesis experiments and molecular dynamics simulations identified additional transient interactions between positively charged residues of the peptides and negatively charged patches distributed on the DMC1 protomers. We propose that BRCA2 and RAD54B stably dock at the P-site of a single DMC1 protomer but also bridge two DMC1 protomers within the presynaptic filament via transient contacts with the adjacent protomer.

biochemistry↗

bioRxiv2025

Generation and characterization of Induced Pluripotent Stem Cell models with SMAD3 mutations to study the molecular mechanism of Loeys-Dietz Syndrome Type III aneurysm formation

Loeys-Dietz Syndrome type 3 (LDS3) is caused by pathogenic (P)/likely pathogenic (LP) variants in the SMAD3 gene and is characterized by aneurysm formation and arterial tortuosity, which can lead to life-threatening complications. There is an unmet need for suitable cell models to study LDS3 at a cellular and molecular level. Induced pluripotent stem (iPS) cells offer a promising approach because they can be genetically modified using CRISPR/Cas9 technology and differentiated into disease-relevant cell types. As it is difficult to obtain aortic vascular smooth muscle cells (VSMCs) from patients, iPS cells differentiated into VSMCs provide an ideal model to study cellular aneurysmal phenotypes. In this study, we generated iPS cell models carrying (P/LP) SMAD3 variants. These cell models were generated either by using CRISPR/Cas9 mediated introduction of indels and deletions to introduce SMAD3 variants, or by reprogramming of fibroblasts derived from SMAD3 patients. These iPS cell lines were characterized for SMAD3 expression by Western blotting and validated for pluripotency through immunofluorescence and qPCR. Moreover, the patient-derived iPS cell lines were shown to differentiate into smooth muscle cells (SMCs), which are relevant to study the molecular mechanisms underlying aneurysm formation in LDS3 patients. Our findings highlight the potential of these iPS-based models to investigate the pathophysiology of LDS3 and facilitate the development of therapeutic strategies for aortic aneurysms.

molecular biology↗

bioRxiv2024

FIGNL1 inhibits homologous recombination in BRCA2 deficient cells by dissociating RAD51 filaments

Homologous recombination (HR) deficiency upon BRCA2 loss arises from defects in the formation of RAD51 nucleoprotein filaments. Here, we demonstrate that loss of the anti-recombinase FIGNL1 retains RAD51 loading at DNA double-stranded breaks (DSBs) in BRCA2-deficient cells, leading to genome stability, HR proficiency, and viability of BRCA2-deficient mouse embryonic stem cells. Mechanistically, we directly show that strand invasion and subsequent HR defects upon BRCA2 loss primarily arises from the unrestricted removal of RAD51 from DSB sites by FIGNL1, rather than from defective RAD51 loading. Furthermore, we identify that the MMS22L-TONSL complex interacts with FIGNL1 and is critical for HR in BRCA2/FIGNL1 double-deficient cells. These findings identify a pathway for tightly regulating RAD51 activity to promote efficient HR, offering insights into mechanisms of chemoresistance in BRCA2-deficient tumors.

genetics↗

bioRxiv2023

Functional analysis of cell lines derived from SMAD3-related Loeys-Dietz Syndrome patients provides insights into genotype-phenotype relations

IntroductionPathogenic (P) and likely pathogenic (LP) variants in the SMAD3 gene cause Loeys-Dietz syndrome type 3 (LDS3), also known as aneurysms-osteoarthritis syndrome (AOS). The phenotype of LDS3 is highly variable and characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with skeletal, cutaneous and facial features. ObjectivesInvestigate the impact of P/LP SMAD3 variants through conducting functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs).The resulting knowledge will optimize interpretation of SMAD3 variants. Material and methodsWe conducted a retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and utilized patient-derived VSMCs to investigate the functional impacts of dominant negative (DN) and haploinsufficient (HI) variants in SMAD3. Additionally, to broaden our cell model accessibility, we performed similar functional analyses on patient-derived fibroblasts carrying SMAD3 variants, differentiating them into myofibroblasts with the same variants. This enabled us to study the functional effects of DN and HI variants in SMAD3 across both patient-derived myofibroblasts and VSMCs. ResultsIndividuals with dominant negative (DN) variants in the MH2 protein interaction domain of SMAD3 exhibited a higher frequency of major events (66.7% vs. 44.0%, p=0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. Moreover, the age at the onset of the first major event was notably younger in individuals with DN variants in MH2, 35.0 years [IQR 29.0-47.0], compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (p=0.065). In functional assays, fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential observed in fibroblasts with HI SMAD3 variants. Additionally, HI SMAD3 variant VSMCs showed elevated SMA expression, while exhibiting altered expression of alternative MYH11 isoforms. Conversely, DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix (ECM) formation compared to control cell lines. These findings collectively indicate distinct functional consequences between DN and HI variants in SMAD3 across fibroblasts and VSMCs, potentially contributing to the observed differences in disease manifestation and age of onset of major events. ConclusionDistinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and evaluating SMA and MYH11 expression. Notably, myofibroblast differentiation seems to be a suitable alternative in vitro test system in comparison to VSMCs. Moreover, there is a notable trend of aortic events occurring at younger age in individuals with a DN SMAD3 variant in the MH2 domain, distinguishing them from those with a DN variant in the MH1 domain or a HI variant.

genetics↗

bioRxiv2023

Chromosome Pairing Through Tensioned DNA Tethers Revealed by BRCA2 Meiotic Domain Deletion

BRCA2 has multiple functional domains that interact with different partners, and is essential for both somatic and meiotic homologous recombination (HR). We created a Brca2{Delta}12-14 mouse model with an internal deletion of the region which we named "the meiotic domain of BRCA2", as its loss results in complete failure of meiotic HR, while somatic HR is intact. The deletion in the protein includes the HSF2BP-binding motifs (exons 12-13) and the DMC1-binding PhePP domain (exon 14). Brca2{Delta}12-14 mice showed complete infertility in both males and females, with sexually dimorphic features. Recombinase foci (both RAD51 and DMC1) were completely undetectable in mutant spermatocytes, but while DMC1 foci were also absent in mutant oocytes, RAD51 foci numbers were only partially reduced. The function of the PhePP domain for meiotic HR is unclear, but both the phenotype of Brca2{Delta}12-14, and our biochemical data indicate that, along with the BRC repeats of BRCA2, PhePP is both critical and specific for DMC1 loading in meiotic HR, analogous to the C-terminal RAD51-specific TR2/CTRB. Further investigation of DSB end processing in Brca2{Delta}12-14 meiocytes and controls, using super-resolution imaging of RPA and SYCP3 led to discovery of two novel features. First, in Brca2{Delta}12-14 oocytes, but not in the spermatocytes nor wild types, we observed RPA foci as doublets [~]200 nm apart, which could represent DSB end resolution into separate nanofoci. Second, we describe RPA structures that are completely HR-dependent and are indicative of long, double-stranded DNA connections between homologs prior to synapsis. Our observations lend support to a model for chromosome alignment via multiple HR-dependent DNA tethers that connect homologs and may be tensed. We propose that tether shortening (e.g. by dynamic adjustment of chromatin loops by meiotic cohesins) provides a plausible molecular mechanism to juxtapose homologs and initiate synapsis. Version 2 Revision SummaryThe main difference compared to version 1 (deposited on October 6 2023) is a more concise and structured description of the tensed DNA tether model of meiotic chromosome pairing, based on the discussions with colleagues and one round of peer review. In the new model presentation, we explicitly separated the inferences from the presented data from the two hypothetical propositions: (1) tether shortening contributes to pairing rather than simply accompanies it, and (2) the apparent tension, which reveals the tethers on chromosome spreads, also exists in the nuclei. We also clarified the definition of the tether, avoiding the ambiguous "RPA tether" term, and provided a more complete overview of the relevant prior literature on proteinaceous bridges and DNA connections. Biochemical data (Fig. 4, S5) has been replicated under uniform conditions and extended to mouse proteins. Manuscript has been reformatted to improve on-screen readability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/561239v2_fig4.gif" ALT="Figure 4"> View larger version (41K): org.highwire.dtl.DTLVardef@1b95b8corg.highwire.dtl.DTLVardef@297466org.highwire.dtl.DTLVardef@198ef4borg.highwire.dtl.DTLVardef@1862be6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO Biochemical characterization of the DMC1-BRCA2 interaction. (A) BRCA2 regions deleted in the mice (yellow) and studied in pull-downs (black), HSF2BP-interacting motifs, PhePP motif and the peptide used in ref. 16 are indicated. Residue numbers for human BRCA2 are shown in black, mouse in blue. (B) GST pull-down with the indicated GST-BRCA2 fragments immobilized on GSH-sepharose beads and used to precipitate recombinant DMC1 or RAD51, followed by immunoblotting with anti-RAD51 antibody (cross-reacts with DMC1) and anti-GST antibodies. Full-size GST-fragment bands are indicated by red arrows, predicted Mw listed below the blot. Experiment was performed twice with same results. (C) Co-precipitation of purified recombinant untagged DMC1 and RAD51 with purified GST-tagged BRCA2 fragment F2s3 (wild type (wt) and F2406A variant (FA)) immobilized on the beads. Bound proteins were analyzed by SDS-PAGE and stained with Coomassie. (D) ITC analysis of the interaction between untagged DMC1 and synthetic peptides corresponding to BRCA2-F2s6 fragment, its F2406A variant, and the peptide from the RAD51-binding TR2 domain, containing a similar FxPP consensus. Experiments were performed twice with the same result, replicates are shown in Figure S5. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/561239v2_figS5.gif" ALT="Figure 5"> View larger version (21K): org.highwire.dtl.DTLVardef@1ac2377org.highwire.dtl.DTLVardef@4c710eorg.highwire.dtl.DTLVardef@924e49org.highwire.dtl.DTLVardef@151a83f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOSupplementary Figure S5.C_FLOATNO (A) Repeat of the ITC experiment shown in Figure 4D. (B) GST pull-down using BRCA2-F2s3, the corresponding mouse BRCA2 fragment (I2327-Q2379), their variants with substitutions in the key phenylalanine (F2406A in human BRCA2 and F2351D in mouse BRCA2, to model the mutation introduced in the previously published mouse strain) and mouse or human his-DMC1. Proteins were expressed in E. coli, precipitated sequentially form crude lysates and detected by immunoblotting with anti-RAD51 and anti-GST antibodies. The experiment was done twice with the same result. C_FIG O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/561239v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1fcff59org.highwire.dtl.DTLVardef@e043a5org.highwire.dtl.DTLVardef@17924dcorg.highwire.dtl.DTLVardef@62fbc3_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

DMC1 and RAD51 bind FxxA and FxPP motifs of BRCA2 via two separate interfaces

In vertebrates, the BRCA2 protein is essential for meiotic and somatic homologous recombination (HR) due to its interaction with RAD51 and DMC1 strand exchange proteins (recombinases). The interaction is mediated by FxxA and FxPP motifs, whose defining feature is the invariant phenylalanine. The FxxA motifs, present in the eight BRC repeats in the central region of BRCA2, compete with the FxxA motif of the linker region of RAD51 that is responsible for recombinase self-oligomerization. In vitro, BRCs disrupt RAD51 nucleoprotein filaments, but they are essential for RAD51 function in the context of the full-length BRCA2 protein. The role of the FxPP motifs is poorly studied but they also contribute to BRCA2 function in cells. In particular, the C-terminal TR2/CTRB domain of BRCA2, which contains an FxPP motif, is required for stabilization of RAD51 filament and replication fork protection. We recently found that deletion of the BRCA2 PhePP domain, which contains another FxPP motif, disrupts DMC1 but not RAD51 function in meiosis. Here we provide a mechanistic explanation for this phenotype by solving the crystal structure of the complex between DMC1 and the PhePP domain of BRCA2. Our structure reveals that, despite sequence similarity, the A-motifs (FxxA) and P-motifs (FxPP) bind to distinct and contiguous sites on the recombinases. The PhePP P-motif binding site is mostly located at the ATPase domain surface of a DMC1 monomer, but also extends to the linker region of the adjacent monomer, thus engaging two adjacent protomers in the DMC1 oligomer. Our structural analysis provides a mechanism explaining how PhePP favors the formation of the DMC1 nucleoprotein filament and stabilizes it. It corroborates and explains the stabilizing effect of the P-motif from BRCA2 TR2/CTRB on RAD51.

biochemistry↗

bioRxiv2023

BRCA2-HSF2BP Oligomeric Ring Disassembly by BRME1 Promotes Homologous Recombination

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks. The HSF2BP-BRME1 complex interacts with BRCA2 to support its function in meiotic HR. In somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here we show that, upon binding to BRCA2, HSF2BP assembles into a large ring-shaped 24-mer consisting of three interlocked octameric rings. Addition of BRME1 leads to dissociation of this ring structure, and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during inter-strand DNA crosslink repair in Xenopus egg extracts. We propose that the control of HSF2BP-BRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting meiotic HR.

biochemistry↗

bioRxiv2023

Distinct mobility patterns of BRCA2 molecules at DNA damage sites

BRCA2 is an essential tumor suppressor protein involved in promoting faithful repair of DNA lesions. The activity of BRCA2 needs to be tuned precisely to be active when and where it is needed. Here, we quantified the spatio-temporal dynamics of BRCA2 in living cells using aberration-corrected multifocal microscopy (acMFM). Using multicolor imaging to identify DNA damage sites, we were able to quantify its dynamic motion patterns in the nucleus and at DNA damage sites. While a large fraction of BRCA2 molecules localized near DNA damage sites appear immobile, an additional fraction of molecules exhibits subdiffusive motion, providing a potential mechanism to retain an increased number of molecules at DNA lesions. Super-resolution microscopy revealed inhomogeneous localization of BRCA2 relative to other DNA repair factors at sites of DNA damage. This suggests the presence of multiple nanoscale compartments in the chromatin surrounding the DNA lesion, which could play an important role in the contribution of BRCA2 to the regulation of the repair process.

cell biology↗

bioRxiv2021

Functional single cell proteomic profiling of cells with abnormal DNA damage response dynamics

Tumor heterogeneity is an important source of cancer therapy resistance. Single cell proteomics has the potential to decipher protein content leading to heterogeneous cellular phenotypes. Single-Cell ProtEomics by Mass Spectrometry (SCoPE-MS) is a recently developed, promising, unbiased proteomic profiling techniques, which allows profiling several tens of single cells for >1000 proteins per cell. However, a method to link single cell proteomes with cellular behaviors is needed to advance this type of profiling technique. Here, we developed a microscopy-based functional single cell proteomic profiling technology, called FUNpro, to link the proteome of individual cells with phenotypes of interest, even if the phenotypes are dynamic or the cells of interest are sparse. FUNpro enables one i) to screen thousands of cells with subcellular resolution and monitor (intra)cellular dynamics using a custom-built microscope, ii) to real-time analyze (intra)cellular dynamics of individual cells using an integrated cell tracking algorithm, iii) to promptly isolate the cells displaying phenotypes of interest, and iv) to single cell proteomically profile the isolated cells. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified PDS5A and PGAM5 proteins contributing to the abnormal DDR dynamics and helping the cells survive after IR.

bioengineering↗

bioRxiv2021

Role of BRCA2 DNA-binding and C-terminal domain on its mobility and conformation in DNA repair

BRCA2 is an essential protein in genome maintenance, homologous recombination and replication fork protection. Its function includes multiple interaction partners and requires timely localization to relevant sites in the nucleus. We investigated the importance of the highly conserved DNA binding domain (DBD) and C-terminal domain (CTD) of BRCA2. We generated BRCA2 variants missing one or both domains in mouse ES cells and defined their contribution in HR function and dynamic localization in the nucleus, by single particle tracking of BRCA2 mobility. Changes in molecular architecture of BRCA2 induced by binding partners of purified BRCA2 was determined by scanning force microscopy. BRCA2 mobility and DNA damage-induced increase in the immobile fraction was largely unaffected by C- terminal deletions. The purified proteins missing CTD and/or DBD were defective in architectural changes correlating with reduced homologous recombination function in cells. These results emphasize BRCA2 activity at sites of damage beyond promoting RAD51 delivery.

molecular biology↗

bioRxiv2021

Experimental evidence for enhanced receptor binding by rapidly spreading SARS-CoV-2 variants

Rapidly spreading new variants of SARS-CoV-2 carry multiple mutations in the viral spike protein which attaches to the angiotensin converting enzyme 2 (ACE2) receptor on host cells. Among these mutations are amino acid changes N501Y (lineage B.1.1.7, first identified in the UK), and the combination N501Y, E484K, K417N (B.1.351, first identified in South Africa), all located at the interface on the receptor binding domain (RBD). We experimentally establish that RBD containing the N501Y mutation results in 9-fold stronger binding to the hACE2 receptor than wild type RBD. The E484K mutation does not significantly influence the affinity for the receptor, while K417N attenuates affinity. As a result, RBD from B.1.351 containing all three mutations binds 3-fold stronger to hACE2 than wild type RBD but 3-fold weaker than N501Y. The recently emerging double mutant E484K/N501Y binds as tight as N501Y. The independent evolution of lineages containing mutations with different effects on receptor binding affinity, viral transmission and immune evasion underscores the importance of global viral genome surveillance and functional characterization.

biochemistry↗

bioRxiv2020

A cryptic BRCA2 repeated motif binds to HSF2BP oligomers with no impact on meiotic recombination

BRCA2 plays a prominent role in meiotic homologous recombination (HR). Loss of BRCA2 or several of its meiotic partners causes fertility defects. One of these partners, HSF2BP, was recently discovered as expressed physiologically in germline and ectopically produced in cancer cells. It has an N-terminal coiled coil motif involved in direct binding to the protein BRME1, and both HSF2BP and BRME1 are essential for meiotic HR during spermatogenesis. It also interacts through its C-terminal Armadillo (ARM) domain with a conserved region of BRCA2 of unknown function. We analyzed the structural properties and functional consequences of the BRCA2-HSF2BP interaction and tested the emerging model of its involvement in meiosis. We solved the crystal structure of the complex between the BRCA2 fragment that is disordered in solution and the HSF2BP dimeric ARM domain. This revealed two previously unrecognized BRCA2 repeats that each interact with one ARM monomer from two different dimers. BRCA2 binding triggers ARM tetramerization, resulting in a complex containing two BRCA2 fragments connecting two ARM dimers. The 3D structures of the BRCA2 repeats are superimposable, revealing conserved contacts between the BRCA2 residues defining the repeats and the HSF2BP residues lining the groove of the ARM. This large interface is responsible for the nanomolar affinity of the interaction, significantly stronger than any other measured interaction involving BRCA2. Deleting exon 12 from Brca2, encoding the first repeat, disrupted BRCA2 binding to HSF2BP in vitro and in cells. However, Brca2{Delta}12/{Delta}12 mice with the same deletion were fertile and did not show any meiotic defects, contrary to the prediction from the model positing that HSF2BP acts as a meiotic localizer of BRCA2. We conclude that the high-affinity interaction between BRCA2 and HSF2BP and the resulting HSF2BP oligomerization are not required for RAD51 and DMC1 recombinase localization to meiotic double strand breaks and for productive meiotic HR.

molecular biology↗

bioRxiv2019

Low Dose Ionizing Radiation Strongly Stimulates Insertional Mutagenesis in a γH2AX Dependent Manner

Extrachromosomal DNA can integrate into the genome with no sequence specificity producing an insertional mutation. This process, which is referred to as random integration (RI), requires a double stranded break (DSB) in the genome. Inducing DSBs by various means, including ionizing radiation, increases the frequency of integration. Here we report that non-lethal physiologically relevant doses of ionizing radiation (10-100 mGy), within the range produced by medical imaging equipment, stimulate RI of transfected and viral episomal DNA in human and mouse cells with an extremely high efficiency. Genetic analysis of stimulated RI (S-RI) revealed that it is distinct from the background RI, requires histone H2AX S139 phosphorylation ({gamma}H2AX) and is not reduced by DNA polymerase {theta} (Polq) inactivation. S-RI efficiency was unaffected by the main DSB repair pathway (homologous recombination and non-homologous end joining) disruptions, but double deficiency in MDC1 and 53BP1 phenocopies {gamma}H2AX inactivation. The robust responsiveness of S-RI to physiological amounts of DSBs has implications for radiation risk assessment and can be exploited for extremely sensitive, macroscopic and direct detection of DSB-induced mutations.

molecular biology↗

bioRxiv2018

The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

biochemistry↗

bioRxiv2018

HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

The tumor suppressor BRCA2 is essential for homologous recombination, replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that a functionally uncharacterized protein, HSF2BP, is involved in a novel, direct and highly evolutionarily conserved interaction with BRCA2. Although HSF2BP was previously described as testis-specific, we find it is expressed in mouse ES cells, in human cancer cell lines, and in tumor samples. Elevated levels of HSF2BP sensitize human cells 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 specifically compromises homologous recombination by preventing BRCA2 and RAD51 loading at the ICL. As increased ectopic expression of HSF2BP occurs naturally, we suggest that it can be considered as a causative agent in FA and a source of cancer-promoting genomic instability.

molecular biology↗