The G2/M checkpoint inhibits mitotic entry upon DNA damage thereby preventing segregation of broken chromosomes and preserving genome stability. The tumor suppressor proteins BRCA1, PALB2 and BRCA2 constitute a BRCA1-PALB2-BRCA2 axis that is essential for homologous recombination (HR)-based DNA double strand break repair. Besides HR, BRCA1 has been implicated in both the initial activation and the maintenance of the G2/M checkpoint, while BRCA2 and PALB2 have been shown to be critical for its maintenance. Here we show that all 3 proteins can play a significant role in both checkpoint activation and checkpoint maintenance, depending on cell type and context, and that PALB2 links BRCA1 and BRCA2 in checkpoint response. The BRCA1-PALB2 interaction can be important for checkpoint activation, whereas the PALB2-BRCA2 complex formation appears to be more critical for checkpoint maintenance. Interestingly, the function of PALB2 in checkpoint response appears to be independent of CHK1 and CHK2 phosphorylation. Following ionizing radiation, cells with disengaged BRCA1-PALB2 interaction show greatly increased chromosomal abnormalities due apparently to combined defects in HR and checkpoint control. These findings provide new insights into DNA damage checkpoint control and further underscore the critical importance of the proper cooperation of the BRCA and PALB2 proteins in genome maintenance.

Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and biolayer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system. Contactin-associated protein-like 2 (CASPR2)6 is a neuronal cell adhesion molecule known in rodents to be necessary for the clustering of the Kv1 potassium channels at juxtaparanodes (1). In myelinated nerves, CASPR2 is confined to the juxtaparanodal region of the axon where it appears to associate with the immunoglobulin domains of TAG-1 (transient axonal glycoprotein-1) to form a scaffold, which clusters the potassium channels Kv1.1 and Kv1.2 (2-4).CASPR2 is predicted to be a type I transmembrane protein of 1331 amino acids with the extracellular domain followed by a single transmembrane domain and a short (48 residues) intracellular domain that terminates with a class II PDZ recognition motif. Computational predictions suggest that CASPR2 has 12 putative N-linked glycosylation sites and 36 Cys residues likely making 18 disulfide bonds, forming 8 independently folded domains: four laminin, neurexin, sex hormone-binding globulin domains (LNS), two epidermal growth factor (EGF) domains, one discoidin domain, and one fibrinogen-like domain (Fig. 1A). CASPR2 shares an overall domain organization with ␣-neurexin-1 despite a relatively low amino acid identity (ϳ23% identity, ϳ39% similarity). However, distinctive features such as a discoidin domain in place of the first LNS domain and a fibrinogen-like domain in place of the 4th LNS domain suggest a different overall structural architecture. No information about the three-dimensional structure of CASPR2, other than that inferred from sequence homology, is currently available. Functionally, only TAG-1 (contactin 2 or CNTN2) has been thus far identified as the extracellular ligand for CASPR2 (2-4).Individuals in a cohort of Amish children, homozygous for a frameshift mutation (single-base G deletion at nucleotide 3709 in exon 22) involving the CNTNAP2 gene, ...

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The BRCA1-PALB2-BRCA2 axis plays an essential role in DNA homologous recombination repair, defect in which drives genome instability and cancer development. How cells with defects in this pathway respond to DNA damage and how tumors develop from these cells remain poorly defined. Here, we analyzed several aspects of the DNA damage response in multiple tissues of-mutant mice in which the interaction between PALB2 and BRCA1 is disengaged. Without any challenge, the mutant mice showed increased endogenous DNA damage. Following ionizing radiation, the mutant mice displayed higher levels of DNA breaks and stronger induction of p53 and p21, but continued DNA synthesis, reduced apoptosis, and accelerated tumor development. The differences in p21 induction, DNA synthesis, and apoptosis between wild-type and mutant mice were substantially more pronounced in the mammary gland than in the intestine, suggesting a potential contributing factor to the increased risk and the tissue specificity of /-associated tumor development. Moreover, the mutant mice showed higher levels of reactive oxygen species and constitutive activation of NF-κB, an antiapoptotic transcription factor inducible by both DNA damage and oxidative stress. Treatment of the mutant mice with an inhibitor of NF-κB reactivated apoptosis and delayed tumor development following radiation. Thus, our results also suggest a prosurvival and pro-oncogenic role of NF-κB in -mutant cells. This study explores novel tumor suppression mechanisms of the BRCA1-PALB2 DNA damage response pathway and implicates NF-κB activation as a protumorogenic event and possible therapeutic target. .