The mechanism(s) for the spatial restriction of histone H2AX phosphorylation and the existence of bona fide
DSBs at persistent γH2AX foci have remained largely unresolved questions (57
). The relative roles of the three PIKKs in modifying substrates in vivo
is not directly addressed here; however, in situ
assays are a novel means to directly probe the mechanisms and biochemical capabilities of PIKKs in a cellular context. Here, we have applied this in situ
biochemical approach to investigate modification of H2AX and other substrates by DNA-PK in chromatin, free of intracellular redundancy for PIKK kinases (15
). Surprisingly, DNA-PK autonomously phosphorylated H2AX and SMC1 on fixed chromatin specifically reconstituting foci at sites that were phosphorylated in vivo
(i.e. sites marked by 53BP1). Introduction of DNA breaks within chromatin or inclusion of exogenous DNA resulted in genome-wide H2AX modification (). These in situ
results indicate that H2AX tails were exposed and suitable DNA-PK substrates throughout chromatin and closely mimic in vivo
observations with hyper-activated ATR kinase (59
). Phosphorylation of H2AX by DNA-PK was neither dependent on the nature of H2AX at break sites nor did it require the localization of other proteins or any active cellular process. In addition, proximity of PIKK substrates to active DNA-PK was not sufficient for phosphorylation, as SMC1 but not ATF2 was modified by DNA-PK at damage sites in chromatin. Collectively, these data reveal an autonomous mechanism by which structural aspects of damaged chromatin and allosteric regulation of DNA-PK can account for localized modification of proteins that are visualized as sub-nuclear foci.
In considering such autonomous activity, we suggest a mechanistic model where DNA DSBs uniquely permit rotational and translational movements in chromatin DNA that cause an immediate local unwinding of chromatin structure at DNA DSBs (D). The nature and extent of this unwinding would be inherent to higher order chromatin organization and structures. The liberated termini would then freely diffuse in a volume dictated by the length of the ‘unwound’ region and its tethering to the larger intact chromatin structure. DNA-PK activity would be limited to H2AX and other proteins/substrates within the volume that the tethered DNA ends could reach. The movement of these DNA termini is by definition proximal to the break and the resulting spatial restriction of DNA-PK activity would result in break-localized protein modification visualized as sub-nuclear foci (D).
Only DNA-PK is directly activated by DNA termini (25
), but break-localized γH2AX foci occur in organisms wholly lacking DNA-PK and in mammalian cells deficient for DNA-PK (18
). Therefore, mechanisms independent of DNA-PK activity and DNA-termini must be functional in localized H2AX phosphorylation in invertebrates and mammals. Considering these facts, we envision a more generalized DNA-damage signaling model. A break in the DNA ‘thread’ releases torsional and translational constraints resulting in a limited unraveling of chromatin. This conformational change in chromatin exposes sequestered allosteric activators and interfaces, including DNA termini. Break-responsive proteins like DNA-PK immediately recognize, bind and/or become activated by association with cognate-binding interfaces and again are spatially restrained by the limited motion of the tethered DNA strands and the extent of unwinding (D). Alternatively, ATM and ATR may function by entirely different mechanisms, and this model may only be relevant to DNA-PK. Regardless, such a mechanism based on chromatin structure would facilitate many parallel, simultaneous and autonomous responses; each queuing on different molecular interfaces, but all immediately, locally and independently respond to DNA breaks.
Much of what is known about mammalian DNA damage responses and relevant proteins is entirely consistent with this proposed mechanism. First, damage foci are formed in cells lacking DNA-PK, ATM, Mre11/Rad50/NBS1 or H2AX itself (6
), indicating independent parallel responses rather than a linear biochemical pathway. Microscopic observations reveal ‘unwinding’ as decondensed 10-nm fibers at repair foci, which may be limited by periodic chromatin anchoring points occurring 5–100 kb apart (65–69
). Consistent with established dimensions, ~10 kb of 10-nm fiber would facilitate a sphere of kinase activity corresponding to a typical γH2AX foci size of 0.25–0.5 um2
, with a corresponding volume containing ~1–2 Mbp of DNA (55
). Furthermore, modification of H2AX based on spatial proximity rather than linear expansion would account for the discontinuities observed in γH2AX along the megabases proximal to DNA breaks (70
). Considering the steric limitations of a DNA terminus, the thousands of individual proteins visualized at ‘damage-induced foci’ are more likely distributed along regions of decondensed chromatin and associated with interfaces other than the terminus itself (D). In fact, binding to chromatin interfaces other than the DNA termini are established for both the 53BP1 and MDC1 proteins that bind methylated histone H4 and γH2AX, respectively (30
). Likewise, the activation of ATM in the absence of DNA termini/breaks and recruitment of proteins to damage sites via protein–protein interactions are entirely consistent with our proposed mechanism (73
Apart from the generalized implications of this mechanism for other kinases, our data show that DNA-PK can autonomously recognize DNA termini in chromatin and become activated to phosphorylate H2AX. The reconstitution of γH2AX foci in fixed cells indicates that break-localized DNA-PK activity is independent of active cellular processes and inherent to DNA-PK and chromatin. We propose that these observations reflect a novel mechanism for break-localized chromatin modification based entirely on the biophysical properties of DNA-PK and chromatin (D). Given the autonomy of this activity, the abundance of DNA-PK, and its affinity for DNA termini (Kd
), it is likely that allosteric activation of DNA-PK at break sites accounts for localized DNA-PK-mediated phosphorylation of proteins in living cells. An additional implication of this model is that DNA-PK bound to a single terminus could actively phosphorylate any number of proximal substrates in cis to initiate DNA-damage signaling, whereas the two termini would have to meet in space to facilitate DNA-PK auto-phosphorylation in trans. Such auto-phosphorylation of DNA-PK facilitates Artemis nuclease activity and brings about a conformational change in DNA-PK important for continued repair by the XRCC4-DNA Ligase IV complex (27
). As a corollary to this line of reasoning, chromatin conformations that limit the meeting of the termini in space would disfavor repair. Although speculative, the residence of persisting DNA breaks in regions of heterochromatin (77
) and the increased size of γH2AX foci at later time points (8
) could be linked phenomena that can be rationalized by our proposed mechanistic model.
Unlike the universal importance of ATM in recognition and signaling of eukaryotic DNA damage, DNA-PK functions are unique to vertebrates. Exactly which aspects of vertebrate DNA repair processes have selected for the addition of DNA-PK remains unclear. These functions may simply be added layers of genome surveillance and repair to safeguard mammalian genomes for decades as opposed to days or months. Alternatively, the added DNA-PK functions may go hand-in-hand with the evolution of more complex chromatin organization that together ensures genetic integrity for decades. In any case, the formation of damage-induced sub-nuclear foci in living cells is independent of ATM, DNA-PK, H2AX or any known single gene product. This parallel redundancy in mammalian DNA repair processes has posed challenges to understanding specific mechanisms of DNA damage responses using traditional methods. Here, we have developed in situ biochemical assays to isolate the mechanism of DNA-PK kinase activity on chromatin substrates. We find that break-localized modification of H2AX in chromatin by DNA-PK relies on allosteric activation by DNA-termini but is otherwise biochemically autonomous. The encoding of DNA damage signaling into higher order chromatin structure could be an elegant means to facilitate immediacy, redundancy, locality and autonomy for the many complex mammalian DNA repair processes. The ability to consider structural and spatial aspects of biochemistry in a cellular context may allow for novel insights into many cellular processes. Further in situ biochemical investigations may prove powerful additions to genetic and molecular techniques in deciphering the spatial and structural components of the complex cellular mechanisms of DNA repair and other cellular processes.