Transient Association of Ku with Nuclear Substrates Characterized Using Fluorescence Photobleaching

Abstract

The autoantigen Ku, composed of subunits Ku70 and Ku86, is necessary for repair of DNA double-strand breaks by nonhomologous end joining. Similarly, Ku participates in repair of DNA double-strand breaks that occur during V(D)J recombination, and it is therefore required for the development of B and T lymphocytes. Although previous studies have identified the DNA-binding activities of Ku, little is known concerning its dynamics, such as the mobility of Ku in the nucleus and its rate of association with substrates. To address this question, fluorescence photobleaching experiments were performed using HeLa cells and B cells expressing a green fluorescent protein (GFP) fusion construct of either Ku70 or Ku86. The results show that Ku moves rapidly throughout the nucleus even following irradiation of the cells. However, the rate of diffusion of Ku was ∼100-fold slower than that predicted from its size. Association of Ku-GFP with a filamentous nuclear structure was also evident, and nuclear extraction experiments suggest that this represents nuclear matrix. A central domain of Ku70 containing its DNA-binding and heterodimerization regions and its nuclear localization signal shows that this alone is sufficient for the observed mobility of Ku70-GFP and its association with nuclear matrix. These data suggest the mobility of Ku is characterized by a transient, high flux association with nuclear substrates that includes both DNA and the nuclear matrix and may represent a mechanism for repair of double-strand breaks using the nuclear matrix as a scaffold.

Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4

“…We found a homogeneous distribution of EGFP-Ku80 throughout the nucleus (except the nucleolus), and FRAP analysis showed free movement of Ku70/80 through the nucleoplasm. This latter observation is markedly different from a recently published study that invokes association of Ku proteins with the nuclear matrix (23). Although the cause for this discrepancy remains to be resolved, it might be explained by different expression levels (we used stable clones, as opposed to transient expression), a different form of the GFP-fusion protein (N-terminal tagging in this study versus C-terminal tagging) and/or a difference in the method of FRAP analysis (we used strip-FRAP instead of spot-FRAP).…”

Cell polarity protein Par3 complexes with DNA-PK via Ku70 and regulates DNA double-strand break repair

“…However, since Par3 is a scaffold protein that plays a key role in establishing cell polarity and TJs, and Ku may serve to tether the free ends of the DSBs to the nuclear matrix [46], it is plausible that Par3 may function as a scaffold to facilitate DNA repair by anchoring the DNA repair apparatus to the broken chromatin. Introduction of DSBs rapidly changes some aspects of higher-order chromatin structure in order to facilitate DNA repair [47][48][49].…”

Visualization of Antigen Presentation by Actin-Mediated Targeting of Glycolipid-Enriched Membrane Domains to the Immune Synapse of B Cell APCs

“…The recovery values were corrected for loss in signal during the experiment as previously described (24,25). The diffusion coefficient (D) of each sample was calculated by fitting the bleaching profile to a Gaussian function (24,26,27).…”

“…Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) measurements were performed as previously described (24), except that in the FRAP experiments the regions were photobleached using a 2-s pulse of laser illumination, and recovery of fluorescence was monitored by collecting frames at a frequency of every 7 s. The mobile fraction of protein and time constant for its recovery were quantitated by fitting the recovery to the function F t /F 0 ϭ F ϱ ϩ F 1 ϫ e (Ϫt/) , where F ϱ represents the fraction of recovery at infinite time and indicates the mobile fraction of the molecule in the bleached region or, inversely, its immobile fraction, and is the time constant for recovery and is inversely proportional to the diffusion coefficient. The recovery values were corrected for loss in signal during the experiment as previously described (24,25). The diffusion coefficient (D) of each sample was calculated by fitting the bleaching profile to a Gaussian function (24,26,27).…”