Functional characterization of a novel Ku70/80 pause site at the H19/Igf2 imprinting control region

The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg(2+), implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.     

One ring to bring them all—The role of Ku in mammalian non-homologous end joining

The repair of DNA double strand breaks is essential for cell survival and several conserved pathways have evolved to ensure their rapid and efficient repair. The non-homologous end joining pathway is initiated when Ku binds to the DNA break site. Ku is an abundant nuclear heterodimer of Ku70 and Ku80 with a toroidal structure that allows the protein to slide over the broken DNA end and bind with high affinity. Once locked into placed, Ku acts as a tool-belt to recruit multiple interacting proteins, forming one or more non-homologous end joining complexes that act in a regulated manner to ensure efficient repair of DNA ends. Here we review the structure and functions of Ku and the proteins with which it interacts during non-homologous end joining.     

Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA.

The Ku protein is composed of two polypeptide subunits, p70 and p80, and binds DNA ends in vitro. Previous studies suggested that p70 and p80 are physically associated in vivo, although such an association may have been mediated by DNA. We have now utilized full-length Ku polypeptides synthesized in vitro to examine the association of p70, p80, and linear DNA to form a complex. In gel filtration chromatography, p70 migrates as a 70-kDa structure, whereas p80 migrates at 150 kDa. Co-translation of the two cDNAs yields complexes which migrate at 300 kDa and contain equimolar quantities of the p70 and p80 polypeptides, providing direct evidence that p70 and p80 assemble into a complex in the absence of DNA. To demonstrate that this recombinant protein complex binds DNA, we developed a radiolabeled protein electrophoretic mobility shift assay. When radiolabeled proteins synthesized in vitro were incubated with linear DNA and fractionated in a nonreducing, nondenaturing gel, a band representing a complex of p70, p80, and the DNA was seen. Formation of this Ku-DNA complex required free DNA ends, and binding to DNA ends was not observed with individual p70 or p80 subunits. DNA binding was not reconstituted by mixing the individual subunits together. These studies thus demonstrate that it is the complex of p70 and p80, not individual p70 or p80, which possesses the DNA binding properties previously described for native Ku protein. These results provide new information about the assembly, structure, and DNA binding properties of the Ku protein.     

DNA依赖蛋白激酶研究进展

DNA依赖蛋白激酶由Ku异二聚体和DNA-PKcs组成,结合Ku蛋白后,DNA-PK激酶活性激活,DNA依赖蛋白激酶具有多功能性,参与DNA修复、基因重组以及复制、转录等多种细胞学过程.     

Disruption of the Arabidopsis AtKu80 gene demonstrates an essential role for AtKu80 protein in efficient repair of DNA double-strand breaks in vivo

Double-strand breaks (DSBs) in DNA may occur spontaneously in the cell or be induced experimentally by gamma-irradiation, and represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination appears to be the major pathway for DSB repair in humans and plants, and it may also be the major route whereby T-DNA integrates into the plant genome during cell transformation. In yeast and mammals, the exposed ends of damaged DNA are bound with high affinity by a dimer of Ku70 and Ku80 proteins, which protects the ends from exonucleases and juxtaposes the two ends of the DSB, independent of sequence homology. Here we report the functional characterization of Ku70 and Ku80 from Arabidopsis thaliana, and demonstrate that AtKu80 and AtKu70 form a heterodimer with DNA binding activity that is specific for DNA ends. An atku80 knockout mutant shows hypersensitivity to the DNA-damaging agents menadione and bleomycin, consistent with a role for AtKu80 in the repair of DSBs in vivo in Arabidopsis.     

Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK

In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.     

Analysis of the mechanism of interaction of simian Ku protein with DNA.

Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes. Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.     

Computational studies on full-length Ku70 with DNA duplexes: base interactions and a helical path

The Ku70/80 heterodimer is among the first responding proteins to recognize and bind the DNA double strand breaks (DSBs). Once Ku is loaded at the DSB, it works as a scaffold to recruit other repair factors in non-homologous end joining thereby facilitates the following repair processes. In this work, we characterized the detailed interactions and binding free energies between a Ku70 subunit and several DNA duplexes, by using some well-established computational methods. The results reveal that the structure of the protein may suffer certain contractions without the company of Ku80, and may experience large conformational changes in the presence of different DNA duplexes. Notably, we observe the closest interactions between Ku70 and DNA can be easily strengthened to form H-bonds with the bases in the minor groove, which is unexpected. However, this finding is supported by the presence of a similar bond between Ku80 and DNA in the published crystal structure (PDB code 1JEY). We suggest that these interactions are responsible for the observed pausing sites when Ku translocates along DNA and the subtle difference in binding with AT- and GC-rich DNA ends. Additionally, simulations indicate the inner surface of the ring encircling the DNA is not flat, but contains a delicate clamp like structure, which is ideal to grip the two strands of DNA in the minor groove and confine the movement of the duplex in a unique helical path.     

Ku must load directly onto the chromosome end in order to mediate its telomeric functions

The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding-defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance.     

DNA-dependent conformational changes in the Ku heterodimer

Ionizing radiation induces DNA double-strand breaks which are repaired by the nonhomologous end joining (NHEJ) pathway. NHEJ is initiated upon Ku binding to the DNA ends and facilitating an interaction with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This heterotrimeric DNA-PK complex is then active as a serine/threonine protein kinase. The molecular mechanisms involved in DNA-PK activation are unknown. Considering the crucial role of Ku in this process, we therefore determined the influence of DNA binding on the structure of the Ku heterodimer. Chemical modification with NHS-biotin and mass spectrometry were used to identify sites of modification. Biotinylation of free Ku revealed several reactive lysines on Ku70 and Ku80 which were reduced or eliminated upon DNA binding. Interestingly, in the predicted C-terminal SAP domain of Ku70, biotinylation patterns were observed which suggest a structural change in this region of the protein induced by DNA binding. Limited proteolytic digests of free and DNA-bound Ku revealed a series of unique peptides, again, indicative of a change in the accessibility of the Ku70 and Ku80 C-terminal domains. A 10 kDa peptide was also identified which was preferentially generated under non-DNA-bound conditions and mapped to the C-terminus of Ku70. These results indicate a DNA-dependent movement or structural change in the C-terminal domains of Ku70 and Ku80 that may contribute to DNA-PKcs binding and activation. These results represent the first demonstration of DNA-induced changes in Ku structure and provide a framework for analysis of DNA-PKcs and the mechanism of DNA-PK activation.     

DNA-dependent protein kinase complex: a multifunctional protein in DNA repair and damage checkpoint

DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase that is activated upon DNA damage generated by ionizing radiation or UV-irradiation. It is a three-protein complex consisting of a 470-kDa catalytic subunit (DNA-PKcs) and the regulatory DNA binding subunits, Ku heterodimer (Ku70 and Ku80). Mouse and human cells deficient in DNA-PKcs are hypersensitive to ionizing radiation and defective in V(D)J recombination, suggesting a role for the kinase in double-strand break repair and recombination. The Ku heterodimer binds to double-strand DNA breaks produced by either DNA damage or recombination, protects DNA ends from degradation, orients DNA ends for re-ligation, and recruits its catalytic subunit and additional factors necessary for successful end-joining. DNA-PK is also involved in an early stage of damage-induced cell cycle arrest, however, it remains unclear how the enzyme senses DNA damage and transmits signals to downstream gene(s) and proteins.     

Loading of the nonhomologous end joining factor, Ku, on protein-occluded DNA ends

The nonhomologous end joining pathway for DNA double strand break repair requires Ku to bind DNA ends and subsequently recruit other nonhomologous end joining factors, including the DNA-dependent protein kinase catalytic subunit and the XRCC4-Ligase IV complex, to the break site. Ku loads at a break by threading the DNA ends through a circular channel in its structure. This binding mechanism explains both the high specificity of Ku for ends and its ability to translocate along DNA once loaded. However, DNA in cells is typically coated with other proteins (e.g. histones), which might be expected to block the ability of Ku to load in this manner. Here we address how the nature of a protein obstruction dictates how Ku interacts with a DNA end. Ku is unable to access the ends within an important intermediate in V(D)J recombination (a complex of RAG proteins bound to cleaved recombination targeting signals), but Ku readily displaces the linker histone, H1, from DNA. Ku also retains physiological affinity for nucleosome-associated ends. Loading onto nucleosome-associated ends still occurs by threading the end through its channel, but rather than displacing the nucleosome, Ku peels as much as 50 bp of DNA away from the histone octamer surface. We suggest a model where Ku utilizes an unusual characteristic of its three-dimensional structure to recognize certain protein-occluded ends without the extensive remodeling of chromatin structure required by other DNA repair pathways.     

Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs

The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK(cs), to that end. The Ku.DNA-PK(cs) complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP(6)) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP(6) bound to DNA-PK(cs) (not to Ku). Here we clearly show that, in fact, IP(6) associates not with DNA-PK(cs), but rather with Ku. Furthermore, the binding of DNA ends and IP(6) to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.