The phospho-dependent role of BRCA2 on the maintenance of chromosome integrity

ABSTRACT

Chromosomal instability is a hallmark of cancer. The tumor suppressor protein BRCA2 performs an important role in the maintenance of genome integrity particularly in interphase; as a mediator of homologous recombination DNA repair pathway, it participates in the repair of DNA double-strand breaks, inter-strand crosslinks and replicative DNA lesions. BRCA2 also protects stalled replication forks from aberrant degradation. Defects in these functions lead to structural chromosomal aberrations. BRCA2 is a large protein containing highly disordered regions that are heavily phosphorylated particularly in mitosis. The functions of these modifications are getting elucidated and reveal emerging activities in chromosome alignment, chromosome segregation and abscission during cell division. Defects in these activities result in numerical chromosomal aberrations. In addition to BRCA2, other factors of the DNA damage response (DDR) participate in mitosis in close association with cell cycle kinases and phosphatases suggesting that the maintenance of genome integrity functions of these factors extends beyond DNA repair. Here we will discuss the regulation of BRCA2 functions through phosphorylation by cell cycle kinases particularly in mitosis, and illustrate with some examples how BRCA2 and other DDR proteins partially rewire their interactions, essentially via phosphorylation, to fulfill mitotic specific functions that ensure chromosome stability.

KEYWORDS:

• Phosphorylation
• chromosomal instability
• BRCA2

Introduction

Inherited mutations in the breast cancer susceptibility gene BRCA2 confer a significant lifetime risk of developing breast, ovarian and with lower penetrance to other cancers including that of the pancreas and the prostate [1,2]. Furthermore, biallelic mutations in BRCA2 are found in a subset of Fanconi anemia patients leading to predisposition to various types of cancer at early age [3]. BRCA2-deficient cells and tumors usually accumulate aberrations in both chromosome structure (translocations, large deletions or chromosome fusions) and number (gains and losses of whole chromosomes) [4,5], illustrating the essential roles of BRCA2 in genome integrity maintenance processes such as DNA repair, DNA replication and cell division.

The most established function of BRCA2 is in homologous recombination (HR); a template-mediated DNA double-strand break (DSB) repair pathway that is considered to be largely error-free [6]. HR is an essential process that uses the redundant genetic information of the sister chromatid (or homologous chromosome) to repair DSBs [7]. The HR repair process starts with the concerted action of the MRN complex and nucleases such as CtIP and EXO1 resecting the DNA-ends, which generates a 3ʹ single-stranded DNA (ssDNA) overhang. The replication protein A (RPA) immediately coats the ssDNA to prevent its degradation and self-annealing [8]. BRCA1, which participates in resection [9], also acts downstream by recruiting other HR factors. In particular, BRCA1 interacts with PALB2 and this complex recruits BRCA2 to the DSB [10], presumably to the dsDNA/ssDNA junction, through the physical interaction of PALB2 WD40 domain and BRCA2 N-terminal region [11,12] (Figure 1a). BRCA2 recruits RAD51 via the BRC repeats located in its central region: as illustrated for BRC4, the BRC motifs fold into two α-helices when interacting with RAD51 (Figure 1b) [13]. Through this interaction, BRCA2 promotes RAD51 assembly and nucleoprotein filament stability in ssDNA, while preventing its nucleation in dsDNA, thus favoring the formation of an active RAD51 nucleoprotein filament and facilitating the displacement of RPA [14]. These activities promote the subsequent steps of strand invasion and homology search in the template DNA strand required for repair [14–19].

Figure 1. Schematic view of BRCA2 protein and some of its interacting partners. (a) BRCA2 is a 3418 aa protein, exhibiting a unique folded domain (PDB 1IYJ [45]). It comprises several binding motifs, here represented by gray rectangles (two of them are displayed in yellow (F1, F2) and further represented in (b, c)). The motifs targeted by kinases and phosphatases are also highlighted. Some of the residues phosphorylated by CDK1/2 and PLK1 are indicated in blue and red, respectively. Docking sites for PLK1 are underlined. Phosphorylation of S3291 is involved in RF protection. Phosphorylation of T77, S193 and T207 are involved in the control of mitotic progression and cytokinesis and indirectly in the HR function of RAD51 (T77). (b) BRCA2 complex involved in HR: Cartoon representation of the BRC4 repeat of BRCA2 (in orange) interacting with the RecA homology domain of RAD51 (in gray) (PDB 1N0W [13]). (c) BRCA2 complex involved in mitosis: Stick representation of BRCA2 peptide from aa 194 to aa 210 (in orange) phosphorylated at T207 (in red) interacting with the Polo-Box domain of PLK1 (cartoon representation in gray) (PDB 6GY2 [42])

The C-terminal DNA-binding domain of BRCA2 (CTD), together with one of its BRC repeats, are sufficient to promote the assembly of RAD51 onto ssDNA and ssDNA/dsDNA junctions [20–22]. A more recent study revealed a second DNA-binding domain in the N-terminal region of BRCA2 (NTD) that can bind different DNA structures, including dsDNA. Interestingly, the NTD per se can promote the DNA strand exchange activity of RAD51 in the presence of RPA in vitro [23]. These findings suggest an interplay between the two BRCA2 DNA-binding domains during HR-mediated transactions. Moreover, increasing evidence suggests that BRCA2 can form a dimer, when free or bound to RAD51 [24–26]. The NTD and CTD of both monomers might contribute to DNA binding during HR.

BRCA2 is also essential in the protection of stalled replication forks (RF), a function that is modulated by phosphorylation by the cyclin-dependent kinases (CDK) 1/2. Following replication stress, RFs are slowed or stalled and, if not protected, newly replicated DNA can undergo unscheduled degradation by nucleases such as MRE11 [27,28]. By using a mutant in the RAD51 binding site located in the extreme C-terminal region of BRCA2 that alters a phosphosite of CDK1/2, it was shown that this region is required to protect nascent DNA strands from aberrant nucleolytic degradation at stalled RF but dispensable for DSB repair [27]. Later studies have demonstrated that BRCA2, as well as BRCA1 and PALB2 and other FA proteins, are required to stabilize the RAD51 filament on the regressed arms of the reversed RF [29–32], a configuration that allows stalled forks to resume replication. In addition, BRCA2 has been proposed to prevent ssDNA gap accumulation under replication stress conditions both at RF junctions and behind forks by stabilizing RAD51 binding [29] or by restraining RF progression [33]. In the absence of BRCA2, forks with persistent ssDNA gaps are potentially converted into reversed forks, leading to extensive degradation.

Several sites have been identified as phosphorylated by the mitotic polo-like kinase 1 (PLK1) in BRCA2 providing the first clues on a possible role of BRCA2 in mitosis [34,35]. In particular, Lin and colleagues showed that PLK1 phosphorylates BRCA2 in mitosis at residue S193 and at least one other residue in the cluster of 203–207. It was later shown that CDK1/2 phosphorylation of T77 in BRCA2 triggers PLK1 docking and phosphorylation of S193 by the same kinase. These events, together with BRCA2 interaction with the actin-binding protein Filamin A, promote BRCA2 localization to the midbody [36] where it interacts with the molecular motor protein Myosin IIC (NMCII) [37]. At the midbody, BRCA2 serves as a scaffold protein helping in the recruitment of endosomal sorting complex required for transport (ESCRT)-associated proteins favoring abscission [36,38]. In addition, the central region of BRCA2 (around BRC5) harbors an interaction site for the kinesin-like coiled-coil high mobility group protein HMG20b/BRAF35. The disruption of the interaction of BRCA2 with any of these factors leads to delayed cell division and binucleated cells [39].

More recently, BRCA2 has been implicated in the regulation of mitotic progression. On the one hand, Lee and colleagues proposed a model in which BRCA2 serves as a platform for P/CAF-mediated acetylation of BUBR1 [35]. This acetylation was proposed to activate the spindle assembly checkpoint (SAC), however, the relevance of BUBR1 acetylation is not well established and whether the interaction between BRCA2 and BUBR1 is direct remains unclear due to confounding results in the literature [40,41]. On the other hand, a more recent study showed that PLK1 phosphorylates BRCA2 at residue T207 which further triggers the binding of PLK1 to phosphorylated BRCA2 facilitating PLK1-mediated phosphorylation of BUBR1. These events promote the stability of kinetochore-microtubules attachments and facilitate chromosome alignment [42]. BRCA2 appears thus as a versatile factor essential for the protection of chromosome integrity, whose interactions are tightly regulated by post-translational modifications.

In the following sections, we provide an overview of the regulation of BRCA2 functions through phosphorylation at several key residues (Table 1). Next, we discuss the consequences that defective phosphorylation of BRCA2 might have on chromosome alignment, stalled RF protection and chromosome segregation. Finally, we describe the role of other DNA repair proteins such as BRCA1 or ATR in mitosis and explore how these factors and BRCA2 might coordinate in this context.

Table 1. Phosphorylation of BRCA2 in G2/M. List of phosphorylation sites is discussed in the text showing BRCA2 phosphorylation site, the responsible kinase, function and consequences in the cell

Phosphorylation site	Kinase	Function	Consequences of phosphorylation
T77	CDK1/2	PLK1 docking and phosphorylation of S193-BRCA2 and S14-RAD51	CK2 phosphorylation of RAD51 and RAD51 interaction with NBS1 (MRN complex) at stressed RF or DSBs^[Citation37,Citation46,Citation54]
S193	PLK1	Localization of BRCA2 at the midbody	Completion of cytokinesis^[37]
T207	PLK1	BRCA2:PLK1:BUBR1:PP2A complex formation at the kinetochore	Faithful chromosome alignment and segregation^[42]
S3291	CDK1/2	Negative regulation of BRCA2-RAD51 interaction	Protection of stalled RF from aberrant degradation^[Citation47,Citation53^]

1. Regulation of BRCA2 functions through phosphorylation

BRCA2 is a large, multi-functional protein that contributes to genomic stability at several levels during the cell cycle. Since several of its functions are cell-cycle dependent, the correct timing for their activation is essential. BRCA2 comprises 3418 amino acids (aa) most of which are disordered [43] as observed by SPOT-Disorder (https://sparks-lab.org/server/spot-disorder/[44]): its unique globular domain comprises about 800 residues [45] (Figure 1a). This feature makes BRCA2 accessible to a large panel of modifying enzymes that regulate its functions [43,45]. Importantly, BRCA2 exhibits a large number of Short LInear Motifs (SLIMs) [36] that are highly conserved from mammals to fishes. Several of these SLIMs are targets for kinases, as inferred from mass spectrometry analyses [35]. These phosphorylation events regulate BRCA2 functions, although how exactly these activities are coordinated during the cell cycle remains poorly understood.

CDK1/2 [12] and PLK1 [13,14] are the main established kinases targeting BRCA2. In 2003, Lin et al. reported that a region comprising aa 1–284 aa of BRCA2 is phosphorylated in mitosis, and suggested that PLK1 is the main kinase responsible for it. The phosphorylated sites belong to a cluster of serines and threonines located around the conserved residue S193 [35] (Figure 1a). Accordingly, combined mutations of S193, T203, S205, S206 and T207 completely abolished phosphorylation of the BRCA2 N-terminal region by PLK1. BRCA2 has also been reported to be phosphorylated by PLK1 during mitosis in the interspace regions located between the BRC repeats [34]. CDK-dependent phosphorylation sites were identified in different regions of BRCA2, further supporting that BRCA2 functions are regulated during the cell cycle [46,47]. In addition, BRCA2 was proposed to be a target of the checkpoint proteins CHK1/2 under DNA-damaging conditions [48], and a target of ataxia telangiectasia mutated and Rad3-related (ATR) [49]. Moreover, BRCA2 contains conserved S/T-Q motifs distributed throughout the sequence, especially enriched between the BRC repeats. The function of these modifications remains to be elucidated.

Phosphorylation events regulating the function of BRCA2 in homologous recombination and the protection of stalled replication forks

Although the phosphorylation of BRCA2 by PLK1 and CDK1/2 was described almost two decades ago [34,35,47], the molecular mechanisms triggered by BRCA2 phosphorylation were, until recently, relatively unclear.

As explained above, the interaction between BRCA2 and RAD51 is essential for both the role of BRCA2 in DNA damage repair by HR and for the protective role of BRCA2 in stalled RF. This interaction is driven via two independent binding sites; the BRC repeats in the central part of the protein and the extreme C-terminal region. The BRC repeats differ from the C-terminus of BRCA2 in that they interact with both monomeric RAD51 and RAD51 nucleoprotein filaments [15], while the C-terminal region only binds to the oligomeric form of RAD51 (Figure 1a, b) [50,51]. The latter is cell-cycle phase specific and negatively regulated through CDK1/2-mediated phosphorylation of serine 3291 (pS3291-BRCA2) [47]. Although the BRC repeats are essential for the function of BRCA2 as a mediator of DSB repair, the C-terminal RAD51 binding site seems dispensable for this activity, whereas it is required for the protection of stalled replication forks from aberrant degradation by nucleases such as MRE11 [27]. This was demonstrated using two separation of function mutants, S3291A and S3291E, that abrogate RAD51 binding by the C-terminus [47]; cells bearing the mutations are defective in replication fork protection but their DSB repair by HR remains essentially intact [27].

In response to replication stress, ATR signaling promotes the interaction between the Hippo pathway kinase LATS1 and CDK2 preventing the phosphorylation of S3291 by CDK2 [52]. This enables BRCA2 interaction with RAD51 filaments, essential for its protective role at the fork [27]. Thus, the phosphorylation of BRCA2 at S3291 by CDK1/2 has been proposed as a molecular switch that regulates RAD51 function at stalled forks and controls entry into mitosis [47,53].

In addition to S3291, CDK1/2 phosphorylates BRCA2 at T77 in late G2-, early M-phase (Figure 1a) [46]. pT77 is the priming event for the interaction between BRCA2 and PLK1, and this interaction facilitates the phosphorylation of S14-RAD51 by PLK1. Such event allows further phosphorylation of RAD51 by CK2 which in turn promotes RAD51 association with the NBS1 component of the MRN complex at induced DNA double-strand breaks or at stalled forks [46,54]. The association of RAD51 and PLK1 is limited to cells bearing mutations in the BRC repeats that alter RAD51 interaction suggesting that BRCA2 binding to RAD51 is important for this S14-RAD51 phosphorylation by PLK1. Thus, the contribution of BRCA2 to the cell response to replication stress is regulated by, at least, two different phosphorylation events initiated by CDK1/2 at both the N- and C-terminus of the protein.

Phosphorylation events regulating the mitotic function of BRCA2

Previous studies have described that the phosphorylation at T77 by CDK also plays an essential role at the end of mitosis, where recruitment of PLK1 favors further phosphorylation of BRCA2 at S193. This second phosphorylation event triggers BRCA2 localization at the midbody and contributes to cytokinesis completion [37]. Consistent with this, alanine substitution of T77 abolishes CDK phosphorylation and thus binding to PLK1 precluding BRCA2 localization at the midbody [37]. A recent work describes a novel role of BRCA2 in mitotic progression. Phosphorylation of T207 by the mitotic kinase PLK1 triggers the binding of PLK1 to pT207 (Figure 1c) and assembly of a tetrameric complex formed by BRCA2, PLK1, BUBR1 and PP2A at the kinetochore [42]. Within this complex, BRCA2 plays the role of a molecular platform that facilitates phosphorylation of its partners critical for proper chromosome alignment.

2. Role of BRCA2 in chromosome alignment and kinetochore-microtubules stability

BUBR1 is involved in two distinct mitotic functions ensuring chromosome stability: it promotes stable kinetochore-microtubule interactions leading to proper chromosome alignment at the metaphase plate and it contributes to the spindle assembly checkpoint (SAC) [55]. BRCA2 has been reported to contribute to the SAC through acetylation of BUBR1 although the mechanism remains poorly understood [41,42]. Recent work provides evidence that BRCA2 is also involved in chromosome alignment [41].

In prometaphase, PLK1 phosphorylates BUBR1 in the tension-sensitive residues S676 and T680 in the KARD motif of BUBR1 favoring the formation of stable kinetochore-microtubule attachments [56]. This activity is tightly regulated by the kinase activity of Aurora B and the phosphatase activity of PP2A [57,58]. Erroneous kinetochore-microtubule interactions are destabilized by Aurora B whereas PP2A counteracts the activity of Aurora B to protect initial attachments. In addition, Aurora B is required for the phosphorylation and localization of BUBR1 at the kinetochores [59]. The activity of PP2A in this context depends on its interaction with BUBR1 via the B56 regulatory subunit, which is favored by the phosphorylation of BUBR1 at the Kinetochore Attachment and Regulatory Domain (KARD) motif (including residues S670, S676 and T680) [57,60–62]. Hence, the interplay between the mitotic proteins PLK1, BUBR1, Aurora B and PP2A in the kinetochores is essential for correct chromosome alignment and faithful chromosome segregation. Recent findings revealed a role for BRCA2 in this interplay; the authors found that T207-BRCA2 is phosphorylated by PLK1 and constitutes a bonafide docking site for this kinase [42]. Moreover, pT207 and the binding of PLK1 to this residue contribute to the formation of a BRCA2:PLK1:BUBR1:PP2A tetrameric complex. Cells expressing BRCA2 variants with a reduced ability to interact with PLK1 at T207 and consequently impaired formation of this tetrameric complex exhibit a high frequency of chromosome misalignment, chromosome segregation errors such as lagging chromosomes, and aneuploidy demonstrating the importance of this complex for chromosome alignment. Over-expression of a phospho-mimic version of BUBR1 (BUBR1-3D; S670D, S676D and T680D) could neither rescue the chromosome misalignment phenotype observed in these cells nor the interaction between BUBR1 and PP2A, suggesting that the docking of PLK1 onto pT207-BRCA2 facilitates the interaction between BUBR1 and PP2A at the kinetochore [42]. Thus, pT207-BRCA2 serves as a docking platform for PLK1 facilitating the interaction between BUBR1 and PP2A. Recruitment of PLK1 to pT207 might favor further phosphorylation of the KARD motif of BUBR1 [37] necessary for proper chromosome alignment, which increases the binding of BUBR1 to the B56 subunit of PP2A. The BUBR1-B56 complex might antagonize phosphorylation of the NDC80 N-terminal tail, which stabilizes the interaction between the kinetochore and the microtubules [57,63]. Furthermore, recruitment of PLK1 at phosphorylated T207 supports further phosphorylation of BRCA2 [37]. In particular, it could increase phosphorylation in the disordered regions located between the BRC repeats, thus regulating the recruitment of the B56 subunit of PP2A (Figure 1a) [42,64].

Altogether, the association between pT207-BRCA2, PLK1, BUBR1 and PP2A is essential for faithful chromosome alignment and segregation.

3. Impact of BRCA2 deficiency on chromosome segregation

As mentioned above, BRCA2 plays a role in replication fork protection, a function that can be uncoupled from its DSB repair activity [27]. This activity prevents aberrant nucleolytic degradation of the newly synthesized strands at stalled replication forks by nucleases such as MRE11 or EXO1, thus preserving genetic information. BRCA2-deficient cells exhibit endogenous replication stress presenting regions of under-replicated DNA [65,66]. Which function of BRCA2, replication fork protection or HR is more important to suppress replication stress and support cell viability is still under debate and it seems to depend on the cellular context [65,67]. The under-replicated DNA is presumably exposed during chromosome condensation, which triggers mitotic DNA synthesis (MiDAS) driven by POLD3 and promoted by MUS81 nuclease among other factors, to complete replication [68]. In BRCA2-deficient cells, this compensatory mitotic DNA synthesis is insufficient, which results in chromosome segregation defects such as chromosome/DNA bridges (Figure 2), ultra-fine bridges (UFB), increased multinucleated cells and DNA lesions in the form of 53BP1 bodies in G1 phase in daughter cells [65,66]. Interestingly, BRCA2 has been shown to support cytokinesis through its localization at the midbody where it serves as a scaffold for several spindle factors such as Filamin A, CEP55 and HMG20b [36,38,39,69]. The consequences of defects in this localization or on the formation of these complexes, as observed in BRCA2-deficient cells and several BRCA2 variants of uncertain clinical significance (VUS) [36], lead to cytokinetic bridges and multinucleated cells (Figure 2); however, the authors did not report increased levels of anaphase/DNA bridges or UFBs in these cells. Thus, defects in replication fork protection or in cytokinesis both hallmarks of BRCA2-deficient cells may lead to similar ultimate outcomes, that is cytokinetic failure and binucleated cells; through presumably different mechanisms, one arising from failed abscission whereas the other resulting from chromosome bridges driven by replication stress.

Figure 2. Schematic view of phosphorylation events regulating BRCA2 function and cell aberrations related to these events. (a) Scheme of BRCA2 showing main phosphorylation by CDK and PLK1 and related interacting partners. BRCA2 is phosphorylated by the kinases CDK (phosphorylation sites in blue) and PLK1 (phosphorylation sites in red) which regulate the interaction between BRCA2 and several of its interaction partners. (b) Scheme showing the role of pT77 and S3291 at DSB/stalled forks. At stalled replication forks, the C-terminal region of BRCA2 interacts with the oligomeric form of RAD51 inhibiting aberrant degradation of the nascent DNA by nucleases like MRE11; phosphorylation by CDK at S3291 negatively regulates this interaction. In response to DSB, CDK phosphorylates BRCA2 at T77 priming PLK1 binding to BRCA2 which favors the phosphorylation of RAD51 by PLK1. PLK1-phosphorylated RAD51 triggers subsequent phosphorylation of RAD51 by CK2 and binding to NBS1 at DSBs [46,54]. A phospho-mimic or phospho-defective mutation at S3291 of BRCA2 leads to unprotected replication forks. Unprotected forks or failure to resume replication of stalled forks, as in the context of BRCA2 loss, result in under replicated DNA leading to anaphase/DNA bridges [65,66,79] as illustrated on the right. (c) Scheme showing the role of pT207 of BRCA2 in mitosis. In mitosis, PLK1 phosphorylates BRCA2 at T207 triggering the formation of the BRCA2:PLK1:BUBR1:PP2A tetrameric complex at the kinetochore important for proper chromosome alignment [42]. Defects in this phosphorylation lead to misaligned and lagging chromosomes due to altered activities of BUBR1 and PP2A at the kinetochore as illustrated on the right. (d) Scheme of the role of pT77 and pS193 in midbody localization and cytokinesis. The phosphorylation of T77 by CDK favors further phosphorylation of S193 by PLK1 allowing BRCA2 localization at the midbody where it

serves as a platform for several proteins regulating cytokinesis and promoting abscission: the motor protein NMCII and the actin-binding protein Filamin A favoring the recruitment of ESCRT-associated proteins Tsg1, CEP55 and Alix [36,37]. Failure of the CDK phosphorylation of T77 and/or phosphorylation of S193-BRCA2 by PLK1 generate cytokinetic aberrations resulting in bi- or multi-nucleated cells [36,37]. NTD, N-terminal DNA-binding domain; CTD, C-terminal DNA-binding domain

In addition to missegregation in the form of chromosome bridges (Figure 2b), cells bearing certain variants of BRCA2 also display misaligned and lagging chromosomes (Figure 2c). These were particularly found in cells bearing VUS S206C and T207A. The origin of these lagging chromosomes may arise, at least in part, from the recently reported function of BRCA2 in promoting the stability of kinetochore-microtubules attachments for proper chromosome alignment [42], as explained above. Surprisingly, although BRCA2-deficient cells display high levels of misaligned chromosomes they do not show significantly increased levels of lagging chromosomes [42]. It is tempting to speculate that the massive number of chromosome bridges arising from the replication stress intrinsic to BRCA2 deficiency somehow prevents the formation of lagging chromosomes. Consistent with this idea, expressing BRCA2 VUS altering chromosome alignment in a BRCA2-deficient background reduced the frequency of chromosome bridges observed in BRCA2-deficient cells while augmenting the number of lagging chromosomes [42].

Although these recent findings suggest an association between chromosome misalignment and an increase in lagging chromosomes, the link between the two remains controversial probably due to the difficulty to track the fate of misaligned chromosomes. In addition, the use of cancer cells versus non-transformed cells seems to modify the outcome [70,71].

Finally, unlike replication stress or cytokinetic failure, a defect in the chromosome alignment function of BRCA2 results in aneuploidy without an increase in the number of tetraploid cells [42].

4. Other DNA repair proteins implicated in mitosis

During mitosis, the kinase activity of Aurora B is essential to destabilize erroneous kinetochore–microtubule interactions [57,58] and dephosphorylation of Aurora B substrates by PP2A prevents premature destabilization of kinetochore-microtubule attachments. In addition, Aurora B is required for the phosphorylation and localization of BUBR1 at the kinetochores [59].

Interestingly, the checkpoint kinase CHK1, which is involved in the response to DNA damage and replication stress in S-phase, stimulates Aurora B catalytic activity in vitro and is required to sustain mitotic arrest in response to the microtubule poison taxol [72]. Accordingly, these authors showed that CHK1 deficient cells display impaired localization of BUBR1 at the kinetochores. More recently, the upstream target of CHK1, ATR, was described to play a DNA-repair independent role in mitosis, which is triggered by centromeric R-loops (DNA-RNA hybrids with a displaced ssDNA loop coated with RPA), resulting from the transcription of centromeric genes [73]. ATR is recruited to the centromeres in prometaphase through the centromeric protein CENP-F and Aurora A. As is the case in the activation of ATR in S-phase [74], R-loops activate ATR localized at the centromeres triggering the phosphorylation of CHK1. This results in a cascade of phosphorylation events that promotes full activation of Aurora B. Compromising this R-loop-driven ATR/CHK1 pathway leads to chromosome segregation errors and aneuploidy [73]. Importantly, this activation of ATR in mitosis is restricted to the kinetochore and does not lead to activation of mitotic CDK1.

To find out how the mitotic and S-phase functions of ATR are coordinated, the Cortez lab used quantitative mass spectrometry in cells depleted by the two known activators of ATR, ETAA1 and TOPBP1, and looked for unique phospho-dependent targets. They showed that the regulation of Aurora B kinase activity in mitosis by ATR is dependent on ETAA1 [75]. This activity of ATR is essential for proper chromosome alignment and a functional spindle assembly checkpoint response.

In addition to the factors mentioned above, Aurora B localization to the kinetochore is dependent on DNA methylation, which is required to maintain centromeric chromatin stability. This DNA methylation is mediated by the DNMT3b methyltransferase. Interestingly, depletion of BRCA1 leads to decreased localization of Aurora B in the kinetochores and this is due to impaired recruitment of DNMT3b to α-satellite repeats, the A-T rich DNA that populates the centromeric region. Consistently, BRCA1 depleted cells show Aurora B mislocalization, display increased chromosome misalignments, segregation errors and elevated levels of aneuploidy [76]. In addition to lagging chromosomes, those cells display chromosome bridges and micronuclei although whether this is due to the role of BRCA1 in interphase or in mitosis requires further study.

Finally, CHK2, a DNA repair kinase generally activated by ATM upon induced DSBs, constitutes another example of DNA repair factor associated with the central spindle and Aurora B activation independently of DNA damage. CHK2 depleted cells delay mitotic progression, deregulate spindle assembly and display misalignment defects. Interestingly, the kinase activity of CHK2 is essential for this function and this activity is driven via the phosphorylation of BRCA1 [77]. It is therefore tempting to speculate that these and other factors that act in concert to preserve genome integrity in interphase, do so as well in the context of mitosis to promote faithful segregation of chromosomes and chromosome stability.

5. Conclusions and perspectives

In summary, it appears that BRCA2 and other key factors that maintain genome integrity in S-phase rewire a part of their interactome in mitosis to localize to the kinetochore and locally control faithful chromosome alignment and segregation to prevent aneuploidy. Further investigations are now needed to fully understand the mechanisms behind the coordination of the distinct functions of these key factors including in the event of DNA damage occurring during mitosis (reviewed in [78]). It is however striking that the functions in DNA repair and mitosis progression of these proteins appear to be tenaciously “uncoupled”. In the case of BRCA2, searching for new phospho-dependent partners and identifying the cellular consequences of mutating the corresponding phosphosites should reveal the molecular switches that control BRCA2 DNA repair and mitotic functions. The use of separation of function mutants and different cell systems will be required to define fundamental aspects of the cellular genome integrity mechanisms such as replication stress tolerance, chromosome alignment, segregation and cell division.

Finally, although the mitotic roles of BRCA2 have not been directly linked to cancer development, an increasing number of variants identified in breast cancer patients alter these functions [36,42]. In addition, BRCA2 mutated tumors display numerical chromosomal aberrations, which are likely to derive from mitotic defects. In the near future, it would be important to compile clinical information on these types of substitution variants to estimate breast cancer risks.

Author contributions

All authors wrote the manuscript; A.E. and S.Z. made the figures. Supervision A.C.

Acknowledgments

We thank Charlotte Martin and Catharina von Nicolai from A. Carreira laboratory for the cell images in figure 2. Current research in A.C. laboratory is funded by the Agence National de Recherche (ANR-17-CE12-0016), Institut National du Cancer (INCa-DGOS_8706), Association pour la Recherche sur le Cancer (ARC), Matmut, Ligue contre le Cancer and the French Cancer Association “Ruban Rose”.

Disclosure statement

The authors declare no competing interests.

Additional information

Funding

This work was supported by the Agence Nationale de la Recherche; Fondation ARC pour la Recherche sur le Cancer; Institut National Du Cancer; Ligue Contre le Cancer and the French Breast Cancer Association “Ruban Rose“.