DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation

Kwon, Youngho; Rösner, Heike I.; Zhao, Weixing; Selemenakis, Platon; He, Zhuoling; Kawale, Ajinkya S.; Katz, Jeffrey N.; Rogers, Cody M.; Neal, Francisco E.; Shabestari, Aida Badamchi; Petrosius, Valdemaras; Singh, Akhilesh K.; Joel, Marina Z.; Holloway, S.; Burma, Sandeep; Mukherjee, Bipasha; Hromas, Robert; Mazin, Alexander V.; Wiese, Claudia; Sørensen, Claus Storgaard; Sung, Patrick

doi:10.1038/s41467-023-36211-x

Cited by 30 publications

(40 citation statements)

References 78 publications

(166 reference statements)

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“…In contrast to the SA point mutation, deletion of the C terminus (∆27) causes HDR defects (Kass et al, 2016;Moynahan et al, 2001), in addition to fork protection and gap suppression defects. The ∆27 deletion includes a KKRR motif which has recently been reported to bind DNA and promote HDR as well as fork protection (Kwon et al, 2023). This motif is intact in the SA mutant.…”

Section: Brca2 Rad51-interacting Carboxyl-terminal Domain Mediates St...mentioning

confidence: 99%

“…Murine BRCA2 protein is 3329 amino acids and interacts with RAD51 at the BRC repeats and at a distinct site in the C terminus that is mutated in BRCA2 S3214A (abbreviated "SA"). BRCA2 ∆27 is a truncation of 190 amino acids that deletes the C-terminal RAD51 interacting site, as well as a recently identified DNA binding motif KKRR (Kwon et al, 2023). B. HDR is not significantly affected in Brca2 SA/SA mouse cells.…”

Section: Limitations Of This Studymentioning

confidence: 99%

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BRCA2 promotes genomic integrity and therapy resistance primarily through its role in homology-directed repair

Lim

Zaman

Jasin

2023

Preprint

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Tumor suppressor BRCA2 functions in homology-directed repair (HDR), protection of stalled replication forks, and suppression of replicative gaps. The relative contributions of these pathways to genome integrity and chemotherapy response are under scrutiny. Here, we report that mouse and human cells require a RAD51 filament stabilization motif in BRCA2 for both fork protection and gap suppression, but not HDR. Loss of fork protection and gap suppression do not compromise genome instability or shorten tumor latency in mice or cause replication stress in human mammary cells. By contrast, HDR deficiency increases spontaneous and replication stress-induced chromosome aberrations and tumor predisposition. Unlike with HDR, fork protection and gap suppression defects are also observed in Brca2 heterozygous mouse cells, likely due to reduced RAD51 stabilization at stalled forks and gaps. Gaps arise from PRIMPOL activity, which is associated with sensitivity to 5-hydroxymethyl-2′-deoxyuridine due to the formation of abasic sites by SMUG1 glycosylase and is exacerbated by poly(ADP-ribose) polymerase inhibition. However, HDR deficiency ultimately modulates sensitivity to chemotherapeutics, including PARP inhibitors.

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Section: Brca2 Rad51-interacting Carboxyl-terminal Domain Mediates St...mentioning

confidence: 99%

Section: Limitations Of This Studymentioning

confidence: 99%

BRCA2 promotes genomic integrity and therapy resistance primarily through its role in homology-directed repair

Lim

Zaman

Jasin

2023

Preprint

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“…Its functional relevance is now supported by the cytological (complete loss of DMC1 foci but partial retention of RAD51 foci, Figures 2-3) and biochemical (details of direct interaction between PhePP and DMC1, Figure 4) data. Our findings suggest that the FxPP consensus does not fully capture the recombinase binding requirements, as PhePP binds DMC1 and not RAD51, while the RAD51-binding “TR2” motif 38 encoded by exon 27 does not bind DMC1, despite similar core sequence (FVPP and FQPP, respectively). BRCA2 thus binds both RAD51 and DMC1 via two distinct domains: the BRC repeats, which bind monomeric forms 46 of both recombinases, and additional FxPP motifs that are recombinase-specific.…”

Section: Discussionmentioning

confidence: 84%

“…We used isothermal titration calorimetry (ITC) to determine that the small synthetic peptide F2s6, corresponding to the conserved region of F2s3, still bound to the purified untagged DMC1 with a Kd of 3.4 µM, and no interaction was observed when the same assay was performed with the F2406A mutant. In addition, testing a similar motif encoded by exon 27, in the so-called TR2 region, revealed that despite the presence of a similar (FQPP) motif, that was shown to bind RAD51 38 , this fragment does not interact with DMC1 (Figure 4E, S5D). Taken together, these results confirm the presence of a specific DMC1-interacting motif in the region encoded by exon 14 of BRCA2.…”

Section: Resultsmentioning

confidence: 99%

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

Koornneef,

van Rossum-Fikkert,

Sleddens-Linkels

et al. 2023

Preprint

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BRCA2 has multiple functional domains that interact with different partners, and is essential for both somatic and meiotic homologous recombination (HR). We created aBrca2Δ12-14mouse 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).Brca2Δ12-14mice 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 (up to 60% fewer, 20% less intense). The relevance of the PhePP domain for meiotic HR has been controversial, but both the phenotype ofBrca2Δ12-14, and our biochemical data indicate that, along with the BRC repeats of BRCA2, PhePP is both essential and specific for DMC1 loading in meiotic HR, analogous to the C-terminal RAD51-specific TR2/CTRB. Further investigation of DSB end processing inBrca2Δ12-14meiocytes and controls, using super-resolution imaging of RPA and SYCP3 led to discovery of two novel features. First, inBrca2Δ12-14oocytes, but not in the spermatocytes nor wild types, we observed RPA foci as doublets ∼200 nm apart, which could represent DSB end separation. Second, we describe RPA structures that are completely HR-dependent and are indicative of long, double-stranded DNA connections between homologs prior to synapsis. The observations led us to a model for chromosome alignment via multiple, tensed DNA tethers that connect the homologous DNA regions. We propose that in combination with dynamic adjustment of chromatin loops by meiotic cohesins, this is a plausible molecular mechanism to bring the homologs closer and initiate synapsis.

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“…It has a stabilizing effect on RAD51-DNA complexes, counteracting BRCs 31,32 . Research on this C-terminal site was focused on S3291, which can be phosphorylated by CDKs at the G2-M transition 28,[30][31][32][33][34][35] .…”

Section: Introductionmentioning

confidence: 99%

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

Miron,

Legrand,

van Rossum-Fikkert

et al. 2023

Preprint

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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.

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DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation

Cited by 30 publications

References 78 publications

BRCA2 promotes genomic integrity and therapy resistance primarily through its role in homology-directed repair

BRCA2 promotes genomic integrity and therapy resistance primarily through its role in homology-directed repair

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

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

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