An autonomous chromatin/DNA-PK mechanism for localized DNA damage signaling in mammalian cells

Abstract

Rapid phosphorylation of histone variant H2AX proximal to DNA breaks is an initiating event and a hallmark of eukaryotic DNA damage responses. Three mammalian kinases are known to phosphorylate H2AX in response to DNA damage. However, the mechanism(s) for damage-localized phosphorylation remains incompletely understood. The DNA-dependent protein kinase (DNA-PK) is the most abundant H2AX-modifying kinases and uniquely activated by binding DNA termini. Here, we have developed a novel approach to examine enzyme activity and substrate properties by executing biochemical assays on intact cellular structures. We apply this approach to examine the mechanisms of localized protein modification in chromatin within fixed cells. DNA-PK retains substrate specificity and independently generates break-localized γH2AX foci in chromatin. In situ DNA-PK activity recapitulates localization and intensity of in vivo H2AX phosphorylation and requires no active cellular processes. Nuclease treatments or addition of exogenous DNA resulted in genome-wide H2AX phosphorylation, showing that DNA termini dictated the locality of H2AX phosphorylation in situ. DNA-PK also reconstituted focal phosphorylation of structural maintenance of chromatin protein 1, but not activating transcription factor 2. Allosteric regulation of DNA-PK by DNA termini protruding from chromatin constitutes an autonomous mechanism for break-localized protein phosphorylation that generates sub-nuclear foci. We discuss generalized implications of this mechanism in localizing mammalian DNA damage responses.

Figure 1.

H2AX serine 139 is biochemically reactive in fixed chromatin. (A) Schematic representation of the in situ biochemical assay on human cells showing cell treatments (left), idealized diagram of DNA damage markers γH2AX and 53BP1 on chromatin through the assay process (right). (B) Primary human fibroblasts (HCA2) were fixed 48 h after X-ray treatments (10 Gy) and either untreated (in vivo) or treated with phosphatases (PPase) and probed for γH2AX and 53BP1 using immunofluorescent techniques; (bottom) 3D plot of γH2AX intensities aforementioned (scale is in arbitrary units). (C) In situ DNA-PK kinase assay showing X-ray-exposed controls (‘In Vivo’), phosphatase (PPase), and DNA-PK kinase reacted (‘In Situ’) cells probed with γH2AX and 53BP1 antibodies as indicated. (D) Representative cells from in situ DNA-PK assays showing relative intensities of γH2AX and 53BP1 immunofluorescent signals. (E) Flat and 3D images of γH2AX and 53BP1 signals from control and in situ kinase cells are shown to reveal co-localization. (F) In situ kinase reactions lacking noted components showing γH2AX and 53BP1 signals.

Figure 2.

DNA-PK activity is spatially constrained by DNA termini. (A) Human fibroblasts were fixed 48 h after mock irradiation (No IR) or at different times following X-ray exposure (10 Gy), then probed with antibodies recognizing either the unphosphorylated H2AX-tail (red) or γH2AX (green). (B) Immunofluorescent H2AX-tail signals were measured from random fields of cells, and average integrated density/nucleus quantified and plotted (n > 25). (C) In situ DNA-PK kinase assays were carried out on dephosphorylated cells pre-treated with buffer (control) micrococcal nuclease, restriction enzyme RsaI or deoxyribonuclease I (DNAse I) as indicated, cells were probed for 53BP1 (red) and γH2AX (green) as noted. (D) γH2AX and 53BP1 immunofluorescent signals were quantified following in situ DNA-PK kinase reactions and noted treatments. The average integrated intensity/area (pixels) of nuclei (n > 20) from random fields was plotted. (E) Dephosphorylated fixed human fibroblasts were reacted with DNA-PK (1.0 pmol) in the presence of exponentially decreasing amounts (10¹ = 625 pmoles) of a 67 bp double-stranded oligonucleotide, then stained for γH2AX (green) and 53BP1 (red). (F) γH2AX and 53BP1 immunofluorescent signals from panels in E were quantified as the average integrated density/nuclei and plotted against the oligonucleotide concentration.

Figure 3.

DNA-PK and Akt1 retain substrate specificity in situ. Fixed primary human fibroblasts either untreated (left panels), subjected to phosphatase treatment (middle panels) or dephosphorlated and reacted with kinases (right panels). Dephosporylated cells were treated with active human (A) DNA-PK or (B) recombinant human Akt1 and probed for phospho-GSK3β (γGSK3β red), γH2AX (green) and counterstained for DNA with DAPI (gray scale) as indicated. Standard in situ kinase reactions were carried out on fixed and dephosphorylated fibroblast cells and probed for (C) phospho-ATF2 (γATF2, red) and 53BP1 (green), or (D) phospho-SMC1 (γSMC1, green) and 53BP1 (red). The two-channel overlays are shown to evaluate localization to DNA damage sites in both cases (merge).

Figure 4.

Persistent DNA damage foci fail to activate the DNA-PK. (A) HCA2 cells were fixed at noted time after X-irradiation (10 Gy) and subjected to in situ DNA-PK kinase assays with reaction products visualized with γH2AX (green) or 53BP1 (red) immunofluorescent staining. (B) Images captured under identical conditions of experiments in panel ‘A’ were plotted in three dimensions to reveal the relative in vivo and in situ γH2AX signal intensities. (C) The area of all of the discrete foci in panel ‘A’ was quantified, and the average foci area in pixels was plotted as a function of time. (D) Mechanistic model for localized H2AX phosphorylation by DNA-PK on chromatin at DNA DSBs. A single locally unwound DNA break is illustrated with translational motion depicted as fading lines within the sphere of DNA-PK kinase activity (green shading). DNA-PK is shown bound to the mobile DNA termini (green circle). Other damage-responsive molecules are represented binding epitopes exposed on chromatin unwinding (red circles, lower strand only). Dimensions of B-form DNA and 10-nm fiber required to span foci radius of 0.25 μM and approximate DNA content of foci volume are indicated (DNA content calculation assumes even DNA distribution within an average human nucleus).