Biology subjects

van Rossum-Fikkert, S. E.

Publications and source records attributed to van Rossum-Fikkert, S. E..

6 recordsLinked to original sources

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↗

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↗

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↗

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↗