The BRCA2 R2645G variant increases DNA binding and induces hyper-recombination.

BRCA2 tumor suppressor protein ensures genome integrity by mediating DNA repair via homologous recombination (HR). This function is executed in part by its canonical DNA binding domain located at the C-terminus (BRCA2CTD), the only folded domain of the protein. Most germline pathogenic missense variants are located in this highly conserved region which binds to single-stranded DNA (ssDNA) and to the acidic protein DSS1. These interactions are essential for the HR function of BRCA2. Here, we report that the variant R2645G, identified in breast cancer and located at the DSS1 interface, unexpectedly increases the ssDNA binding activity of BRCA2CTDin vitro. Human cells expressing this variant display a hyper-recombination phenotype, chromosomal instability in the form of chromatid gaps when exposed to DNA damage, and increased PARP inhibitor sensitivity. In mouse embryonic stem cells (mES), this variant alters viability and confers sensitivity to cisplatin and Mitomycin C. These results suggest that BRCA2 interaction with ssDNA needs to be tightly regulated to limit HR and prevent chromosomal instability and we propose that this control mechanism involves DSS1. Given that several missense variants located within this region have been identified in breast cancer patients, these findings might have clinical implications for carriers.

Dynamics of Rad9 chromatin binding and checkpoint function are mediated by its dimerization and are cell cycle-regulated by CDK1 activity.

Saccharomyces cerevisiae Rad9 is required for an effective DNA damage response throughout the cell cycle. Assembly of Rad9 on chromatin after DNA damage is promoted by histone modifications that create docking sites for Rad9 recruitment, allowing checkpoint activation. Rad53 phosphorylation is also dependent upon BRCT-directed Rad9 oligomerization; however, the crosstalk between these molecular determinants and their functional significance are poorly understood. Here we report that, in the G1 and M phases of the cell cycle, both constitutive and DNA damage-dependent Rad9 chromatin association require its BRCT domains. In G1 cells, GST or FKBP dimerization motifs can substitute to the BRCT domains for Rad9 chromatin binding and checkpoint function. Conversely, forced Rad9 dimerization in M phase fails to promote its recruitment onto DNA, although it supports Rad9 checkpoint function. In fact, a parallel pathway, independent on histone modifications and governed by CDK1 activity, allows checkpoint activation in the absence of Rad9 chromatin binding. CDK1-dependent phosphorylation of Rad9 on Ser11 leads to specific interaction with Dpb11, allowing Rad53 activation and bypassing the requirement for the histone branch.

Haspin Modulates the G2/M Transition Delay in Response to Polarization Failures in Budding Yeast.

Symmetry breaking by cellular polarization is an exquisite requirement for the cell-cycle of Saccharomyces cerevisiae cells, as it allows bud emergence and growth. This process is based on the formation of polarity clusters at the incipient bud site, first, and the bud tip later in the cell-cycle, that overall promote bud emission and growth. Given the extreme relevance of this process, a surveillance mechanism, known as the morphogenesis checkpoint, has evolved to coordinate the formation of the bud and cell cycle progression, delaying mitosis in the presence of morphogenetic problems. The atypical protein kinase haspin is responsible for histone H3-T3 phosphorylation and, in yeast, for resolution of polarity clusters in mitosis. Here, we report a novel role for haspin in the regulation of the morphogenesis checkpoint in response to polarity insults. Particularly, we show that cells lacking the haspin ortholog Alk1 fail to achieve sustained checkpoint activation and enter mitosis even in the absence of a bud. In alk1Δ cells, we report a reduced phosphorylation of Cdc28-Y19, which stems from a premature activation of the Mih1 phosphatase. Overall, the data presented in this work define yeast haspin as a novel regulator of the morphogenesis checkpoint in Saccharomyces cerevisiae, where it monitors polarity establishment and it couples bud emergence to the G2/M cell cycle transition.

Haspin regulates Ras localization to promote Cdc24-driven mitotic depolarization.

Cell polarization is of paramount importance for proliferation, differentiation, development, and it is altered during carcinogenesis. Polarization is a reversible process controlled by positive and negative feedback loops. How polarized factors are redistributed is not fully understood and is the focus of this work. In Saccharomyces cerevisiae, mutants defective in haspin kinase exhibit stably polarized landmarks and are sensitive to mitotic delays. Here, we report a new critical role for haspin in polarisome dispersion; failure to redistribute polarity factors, in turn, leads to nuclear segregation defects and cell lethality. We identified a mitotic role for GTP-Ras in regulating the local activation of the Cdc42 GTPase, resulting in its dispersal from the bud tip to a homogeneous distribution over the plasma membrane. GTP-Ras2 physically interacts with Cdc24 regulateing its mitotic distribution. Haspin is shown to promote a mitotic shift from a bud tip-favored to a homogenous PM fusion of Ras-containing vesicles. In absence of haspin, active Ras is not redistributed from the bud tip; Cdc24 remains hyperpolarized promoting the activity of Cdc42 at the bud tip, and the polarisome fails to disperse leading to erroneously positioned mitotic spindle, defective nuclear segregation, and cell death after mitotic delays. These findings describe new functions for key factors that modulate cell polarization and mitotic events, critical processes involved in development and tumorigenesis.

Kar9 controls the nucleocytoplasmic distribution of yeast EB1.

The precise temporal and spatial concentration of microtubule-associated proteins (MAPs) within the cell is fundamental to ensure chromosome segregation and correct spindle positioning. MAPs form an intricate web of interactions among each other and compete for binding sites on microtubules. Therefore, when assessing cellular phenotypes upon MAP up- or downregulation, it is important to consider the protein interaction network between individual MAPs. Here, we show that changes in the amounts of the spindle positioning factor Kar9 specifically affect the distribution of yeast EB1 on spindle microtubules, without influencing other microtubule-associated interacting partners of Kar9, i.e. yeast XMAP215 and CLIP-170. Alterations in the distribution of yeast EB1 explain chromosome segregation defects upon knockout, overexpression or stabilization of Kar9 and provide an example for non-linear effects on MAP behavior after perturbation of their equilibrium.

Regulation of a Spindle Positioning Factor at Kinetochores by SUMO-Targeted Ubiquitin Ligases.

Correct function of the mitotic spindle requires balanced interplay of kinetochore and astral microtubules that mediate chromosome segregation and spindle positioning, respectively. Errors therein can cause severe defects ranging from aneuploidy to developmental disorders. Here, we describe a protein degradation pathway that functionally links astral microtubules to kinetochores via regulation of a microtubule-associated factor. We show that the yeast spindle positioning protein Kar9 localizes not only to astral but also to kinetochore microtubules, where it becomes targeted for proteasomal degradation by the SUMO-targeted ubiquitin ligases (STUbLs) Slx5-Slx8. Intriguingly, this process does not depend on preceding sumoylation of Kar9 but rather requires SUMO-dependent recruitment of STUbLs to kinetochores. Failure to degrade Kar9 leads to defects in both chromosome segregation and spindle positioning. We propose that kinetochores serve as platforms to recruit STUbLs in a SUMO-dependent manner in order to ensure correct spindle function by regulating levels of microtubule-associated proteins.

To trim or not to trim: progression and control of DSB end resection.

DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, since they can lead to genome instability and chromosome rearrangements, which are hallmarks of cancer cells. To face this kind of lesion, eukaryotic cells developed two alternative repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Repair pathway choice is influenced by the cell cycle phase and depends upon the 5'-3' nucleolytic processing of the break ends, since the generation of ssDNA tails strongly stimulates HR and inhibits NHEJ. A large amount of work has elucidated the key components of the DSBs repair machinery and how this crucial process is finely regulated. The emerging view suggests that besides endo/exonucleases and helicases activities required for end resection, molecular barrier factors are specifically loaded in the proximity of the break, where they physically or functionally limit DNA degradation, preventing excessive accumulation of ssDNA, which could be threatening for cell survival.

Yeast haspin kinase regulates polarity cues necessary for mitotic spindle positioning and is required to tolerate mitotic arrest.

Haspin is an atypical protein kinase that in several organisms phosphorylates histone H3Thr3 and is involved in chromosome segregation. In Saccharomyces cerevisiae, H3Thr3 phosphorylation has never been observed and the function of haspin is unknown. We show that deletion of ALK1 and ALK2 haspin paralogs causes the mislocalization of polarisome components. Following a transient mitotic arrest, this leads to an overly polarized actin distribution in the bud where the mitotic spindle is pulled. Here it elongates, generating anucleated mothers and binucleated daughters. Reducing the intensity of the bud-directed pulling forces partially restores proper cell division. We propose that haspin controls the localization of polarity cues to preserve the coordination between polarization and the cell cycle and to tolerate transient mitotic arrests. The evolutionary conservation of haspin and of the polarization mechanisms suggests that this function of haspin is likely shared with other eukaryotes, in which haspin may regulate asymmetric cell division.