Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.

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The means by which the cell regulates protein kinase CK2 remain obscure. However, natural polyamines, cellular compounds required for cell proliferation, have been reported to strongly stimulate CK2-mediated phosphorylation of a number of substrates. Using spermine analogs, we have shown that polyamines directly interact with the CK2 ␤ subunit, and the chemical features of the highly acidic binding site (Asp 51 -Tyr 80 ) have been determined. In the present study, we show that the isolated ␤ subunit region extending from residue Asp 51 to Pro 110 exhibits a specific and efficient polyamine binding activity similar to that of the entire ␤ subunit. Moreover, the replacement of Glu 60 , Glu 61 , and Glu 63 of the ␤ subunit by 3 alanine residues leads to a loss of the spermine-induced stimulation of CK2 activity which correlates with a decrease in spermine binding affinity. Thermal stability studies indicate that the binding of spermine induces a 4°C decrease of the T m value for the holoenzyme. This was confirmed by circular dichroism analyses, which show that the 6°C negative shift of the CK2 T m value provoked by spermine binding, reflects a conformational change in the kinase. Together, these observations strongly suggest that this newly defined polyamine-binding domain is involved in the intrasteric regulation of CK2 activity.Protein kinase CK2 (CK2) 1 is a serine/threonine protein kinase present in the cell cytoplasm and nucleus of eukaryotic organisms from yeast to man (1, 2). The kinase is made of the association of three dissimilar subunits, i.e. the catalytic subunits ␣ and ␣Ј of 35-44 kDa (3) and the ␤ subunit of 24 -29 kDa, to generate native structures exhibiting the stoichiometry ␣ 2 ␤ 2 , ␣Ј 2 ␤ 2 , and ␣␣Ј␤ 2 . The ␤ subunit contains two autophosphorylation sites located at Ser 2 and Ser 3 and is termed the regulatory subunit because of its potential to stimulate the activity of the ␣ subunit in the tetramer by 5-10-fold (4 -6). Among the numerous substrates of CK2, a large number corresponds to transcription factors and oncoproteins such as Myc (7), Myb (2, 8), Fos (9), and the anti-oncogene p53 (10). Other proteins localized in the cytoplasm or associated to membranes were also identified as CK2 substrates. The disruption of the genes encoding the CK2 catalytic subunits ␣ and ␣Ј led to a lethal phenotype in yeasts, underscoring CK2's essential role in cell proliferation (11).To date, no intracellular messenger has been characterized as a crucial regulator of CK2. However, in vitro experiments that show significant stimulation of the CK2 catalytic activity in the presence of naturally occurring polyamines (12), provides strong evidence that such regulation exists. The stimulation by polyamines is preferentially observed with selected substrates, such as casein or the transcription factor MyoD, and is strictly dependent on the presence of the ␤ subunit (13) and on low magnesium concentrations (14). Further evidence supporting a role for polyamines as physiological CK2 stimulators includes the observa...