Methylation of the central transcriptional regulator KLF4 by PRMT5 is required for DNA end resection and recombination.

Cell fitness and survival upon exposure to DNA damage depends on the repair of DNA lesions. Interestingly, cellular identity does affect and finetunes such response, although the molecular basis of such differences between tissues and cell types is not well understood. Thus, a possibility is that DNA repair itself is controlled by the mechanisms that govern cell identity. Here we show that the KLF4, involved in cellular homeostasis, proliferation, cell reprogramming and cancer development, directly regulates resection and homologous recombination proficiency. Indeed, resection efficiency follows KLF4 protein levels, i.e. decreases upon KLF4 downregulation and increases when is overexpressed. Moreover, KLF4 role in resection requires its methylation by the methyl-transferase PRMT5. Thus, PRMT5 depletion not only mimics KLF4 downregulation, but also showed an epistatic genetic relationship. Our data support a model in which the methylation of KLF4 by PRMT5 is a priming event required to license DNA resection and homologous recombination.

DNA end resection requires constitutive sumoylation of CtIP by CBX4.

DNA breaks are complex DNA lesions that can be repaired by two alternative mechanisms: non-homologous end-joining and homologous recombination. The decision between them depends on the activation of the DNA resection machinery, which blocks non-homologous end-joining and stimulates recombination. On the other hand, post-translational modifications play a critical role in DNA repair. We have found that the SUMO E3 ligase CBX4 controls resection through the key factor CtIP. Indeed, CBX4 depletion impairs CtIP constitutive sumoylation and DNA end processing. Importantly, mutating lysine 896 in CtIP recapitulates the CBX4-depletion phenotype, blocks homologous recombination and increases genomic instability. Artificial fusion of CtIP and SUMO suppresses the effects of both the non-sumoylatable CtIP mutant and CBX4 depletion. Mechanistically, CtIP sumoylation is essential for its recruitment to damaged DNA. In summary, sumoylation of CtIP at lysine 896 defines a subpopulation of the protein that is involved in DNA resection and recombination.The choice between non-homologous end-joining and homologous recombination to repair a DNA double-strand break depends on activation of the end resection machinery. Here the authors show that SUMO E3 ligase CBX4 sumoylates subpopulation of CtIP to regulate recruitment to breaks and resection.

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice.

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.

Mechanism for astral microtubule capture by cortical Bud6p priming spindle polarity in S. cerevisiae.

BACKGROUND: Budding yeast is a unique model to dissect spindle orientation in a cell dividing asymmetrically. In yeast, this process begins with the capture of pole-derived astral microtubules (MTs) by the polarity determinant Bud6p at the cortex of the bud in G(1). Bud6p couples MT growth and shrinkage with spindle pole movement relative to the contact site. This activity resides in N-terminal sequences away from a domain linked to actin organization. Kip3p (kinesin-8), a MT depolymerase, may be implicated, but other molecular details are essentially unknown. RESULTS: We show that Bud6p and Kip3p play antagonistic roles in controlling the length of MTs contacting the bud. The stabilizing role of Bud6p required the plus-end-tracking protein Bim1p (yeast EB1). Bim1p bound Bud6p N terminus, an interaction that proved essential for cortical capture of MTs in vivo. Moreover, Bud6p influenced Kip3p dynamic distribution through its effect on MT stability during cortical contacts via Bim1p. Coupling between Kip3p-driven depolymerization and shrinkage at the cell cortex required Bud6p, Bim1p, and dynein, a minus-end-directed motor helping tether the receding plus ends to the cell cortex. Validating these findings, live imaging of the interplay between dynein and Kip3p demonstrated that both motors decorated single astral MTs with dynein persisting at the plus end in association with the site of cortical contact during shrinkage at the cell cortex. CONCLUSIONS: Astral MT shrinkage linked to Bud6p involves its direct interaction with Bim1p and the concerted action of two MT motors-Kip3p and dynein.