Atomic force microscopy of DNA molecules stretched by spin-coating technique

We have developed an effective approach to stretching DNA molecules with the flow of fluid generated by spin coating. Well-stretched A DNA molecules were observed using atomic force microscopy. Substrate properties sensitively affected the stretching behavior of DNA. Our experimental findings revealed that a mica surface treated with crystal violet, a cationic dye molecule, is suitable to the spin-coating procedure for stretching DNA. Moreover, compared with relaxed DNA, we observed reduced height of the stretched DNA, which was attributed mainly to elongation force applied to the DNA molecules from the fluid flow and strong adhesion force between DNA and the substrate. This simple and effective method for preparing stretched DNA could be useful in physically mapping genomic DNA in a high throughput.     

Purification and identification of positive regulators binding to a novel element in the c-Jun promoter

A putative response element, GAGCCTC, was observed years ago in footprinting analysis of the c-jun promoter, and here we investigate its function in regulating c-jun expression and identify a protein complex that binds there. Electrophoretic mobility shift assays demonstrate a sequence-specific binding complex with this element in HEK293 cells. Additionally, unlabeled consensus AP-1 element DNA, but not a similar NF-jun element DNA, competes with complex formation. Mutations of this element decrease c-jun promoter reporter activity by nearly 5-fold in HEK293 cells. A new, two-step oligonucleotide trapping technique was developed to purify the element binding proteins. LC-nanospray-ESI-MS/MS identification and Western blotting show that the purified complex contains Ku80 and c-jun, which was further confirmed by antibody supershift, by immunoprecipitation with Southwestern blot or with UV cross-linking analysis in vitro as well as chromatin immunoprecipitation in vivo. c-Jun promoter activity and c-jun expression were decreased by Ku80 siRNA introduction. A mutant Ku80 plasmid with normal amino acid sequence but immune to the siRNA recovers c-jun promoter activity from siRNA inhibition. Similarly, Ku70 wild type transfection can also upregulate c-jun promoter activity. Thus, Ku80-c-jun activates c-jun expression by binding to this GAGCCTC element in the c-jun promoter and Ku70 may also serve a role.     

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.     

2-Step purification of the Ku DNA repair protein expressed in Escherichia coli

The Ku protein is involved in DNA double-strand break repair by non-homologous end-joining (NHEJ), which is crucial to the maintenance of genomic integrity in mammals. To study the role of Ku in NHEJ we developed a bicistronic Escherichia coli expression system for the Ku70 and Ku80 subunits. Association of the Ku70 and Ku80 subunits buries a substantial amount of surface area (approximately 9000 A2 [J.R. Walker, R.A. Corpina, J. Goldberg, Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair, Nature 412 (2001) 607-614]), which suggests that herterodimerization may be important for protein stability. N-terminally His6-tagged Ku80 was soluble in the presence, but not in the absence, of bicistronically expressed untagged Ku70. In a 2-step purification, metal chelating affinity chromatography was followed by step-gradient elution from heparin-agarose. Co-purification of equimolar amounts of His6-tagged Ku80 and untagged Ku70 was observed, which indicated heterodimerization. Recombinant Ku bound dsDNA, activated the catalytic subunit of the DNA-dependent kinase (DNA-PKcs) and functioned in NHEJ reactions in vitro. Our results demonstrate that while the heterodimeric interface of Ku is extensive it is nonetheless possible to produce biologically active Ku protein in E. coli.     

Kinetic analysis of the Ku-DNA binding activity reveals a redox-dependent alteration in protein structure that stimulates dissociation of the Ku-DNA complex

Ku is a heterodimeric protein comprising 70- and 80-kDa subunits that participate in the non-homologous end-joining (NHEJ) repair pathway for rejoining DNA double strand breaks. We have analyzed the pre-steady state binding of Ku with various DNA duplex substrates and identified a redox-sensitive Ku-DNA interaction. Pre-steady state analysis of Ku DNA binding was monitored via intrinsic Ku quenching upon binding DNA and revealed that, under fully reduced conditions, binding occurred in a single-step process. Reactions performed under limited reduction revealed a two-step binding process, whereas under fully oxidized conditions, we were unable to detect quenching of Ku fluorescence upon binding DNA. The differential quenching observed under the different redox conditions could not be attributed to two Ku molecules binding to a single substrate or Ku sliding inward on the substrate. Although only modest differences in Ku DNA binding activity were observed in the stoichiometric anisotropy and electrophoretic mobility shift assay studies, as a function of redox conditions, a dramatic difference in the rate of Ku dissociation from DNA was observed. This effect was also induced by diamide treatment of Ku and could be abrogated by dithiothreitol treatment, demonstrating a reversible redox effect on the stability of the Ku-DNA complex. The redox-dependent alteration in Ku-DNA interactions is manifested by a redox-dependent alteration in Ku structure, which was confirmed by limited proteolysis and mass spectrometry analyses. The results support a model for the interaction of Ku with DNA that is regulated by redox status and is achieved by altering the dissociation of the Ku-DNA complex.     

Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair

DNA-PKcs is a large (approximately 470 kDa) kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). DNA-PKcs is recruited to DSBs by the Ku70/Ku80 heterodimer, with which it forms the core of a multiprotein complex that promotes synapsis of the broken DNA ends. We have purified the human DNA-PKcs/Ku70/Ku80 holoenzyme assembled on a DNA molecule. Its three-dimensional (3D) structure at approximately 25 Angstroms resolution was determined by single-particle electron microscopy. Binding of Ku and DNA elicits conformational changes in the FAT and FATC domains of DNA-PKcs. Dimeric particles are observed in which two DNA-PKcs/Ku70/Ku80 holoenzymes interact through the N-terminal HEAT repeats. The proximity of the dimer contacts to the likely positions of the DNA ends suggests that these represent synaptic complexes that maintain broken DNA ends in proximity and provide a platform for access of the various enzymes required for end processing and ligation.     

Involvement of DNA End-Binding Protein Ku in Ty Element Retrotransposition

Saccharomyces cerevisiae Ty elements are retrotransposons whose life cycles are strikingly similar to those of retroviruses. They transpose via an RNA intermediate that is converted to linear double-stranded cDNA and then inserted into the host genome. Although Ty integration is mediated by the element-encoded integrase, it has been proposed that host factors are involved in this process. Here, we show that the DNA end-binding protein Ku, which functions in DNA double-strand break repair, potentiates retrotransposition. Specifically, by using a galactose-inducible Ty1 system, we found that in vivo, Ty1 retrotransposition rates were substantially reduced in the absence of Ku. In contrast, this phenotype was not observed with yeast strains containing mutations in other genes that are involved in DNA repair. We present evidence that Ku associates with Ty1 viruslike particles both in vitro and in vivo. These results provide an additional role for Ku and suggest that it might function in the life cycles of retroelements in other systems.     

Involvement of the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis

Ku70 plays an important role in DNA damage repair and prevention of cell death. Previously, we reported that apoptosis caused a decrease in cellular Ku70 levels. In this study, we analyzed the mechanism of how Ku70 levels decrease during drug-induced apoptosis. In HeLa cells, staurosporin (STS) caused a decrease in Ku70 levels without significantly affecting Ku70 mRNA levels. We found that Ku70 protein was highly ubiquitinated in various cell types, such as HeLa, HEK293T, Dami (a megakaryocytic cell line), endothelial, and rat kidney cells. An increase in ubiquitinated Ku70 protein was observed in apoptotic cells, and proteasome inhibitors attenuated the decrease in Ku70 levels in apoptotic cells. These results suggest that the ubiquitin-proteasome proteolytic pathway plays a role in decreasing Ku70 levels in apoptotic cells. Ku70 forms a heterodimer with Ku80, which is required for the DNA repair activity of Ku proteins. We also found that Ku80 levels decreased in apoptotic cells and that Ku80 is a target of ubiquitin. Ubiquitinated Ku70 was not found in the Ku70-Ku80 heterodimer, suggesting that modification by ubiquitin inhibits Ku heterodimer formation. We propose that the ubiquitin-dependent modification of Ku70 plays an important role in the control of cellular levels of Ku70.     

Displacement of DNA-PKcs from DNA ends by the Werner syndrome protein

The DNA-dependent protein kinase (DNA-PK) complex, which is composed of a DNA-dependent kinase subunit (DNA-PKcs) and the Ku70/80 heterodimer, is involved in DNA double-strand break repair by non-homologous end joining (NHEJ). Ku70/80 interacts with the Werner syndrome protein (WRN) and stimulates WRN exonuclease activity. To investigate a possible function of WRN in NHEJ, we have examined the relationship between DNA-PKcs, Ku and WRN. First, we showed that WRN forms a complex with DNA-PKcs and Ku in solution. Next, we determined whether this complex assembles on DNA ends. Interestingly, the addition of WRN to a Ku:DNA-PKcs:DNA complex results in the displacement of DNA-PKcs from the DNA, indicating that the triple complex WRN:Ku:DNA-PKcs cannot form on DNA ends. The displacement of DNA-PKcs from DNA requires the N- and C-terminal regions of WRN, both of which make direct contact with the Ku70/80 heterodimer. Moreover, exonuclease assays indicate that DNA-PKcs does not protect DNA from the nucleolytic action of WRN. These results suggest that WRN may influence the mechanism by which DNA ends are processed.     

Purification and characterization of Ku-2, an octamer-binding protein related to the autoantigen Ku

The octamer motif (ATTTGCAT) is an important regulatory element in eukaryotic gene expression. A previously unidentified protein that recognizes this motif has been isolated from the human B cell line, Daudi. The protein, which we term Ku-2, bears a close resemblance to the DNA-binding autoantigen Ku. Like Ku, it is a heterodimer with subunits of 83 and 72 kDa; antisera raised against either subunit of Ku cross-react with Ku-2. Two peptides have been sequenced and show a strong similarity to regions in the corresponding subunits of Ku. The sequences are not identical, however, suggesting that Ku-2 may be a B cell homologue of Ku. Both Ku and Ku-2 bind to the termini of DNA duplexes, but Ku-2 also binds to an internal octamer motif. It is not known whether Ku shares the latter property or whether the octamer binding is a consequence of sequence differences between the two proteins. Ku-2 does not react with antisera against the POU domain of the octamer-binding protein Oct-2, indicating that the DNA binding domains of the two proteins are dissimilar despite the ability of both to bind to the octamer motif. We discuss the evidence for the existence of a family of octamer-binding proteins related to Ku.     

Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs

Recognition of DNA double-strand breaks during non-homologous end joining is carried out by the Ku70-Ku80 protein, a 150 kDa heterodimer that recruits the DNA repair kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the lesion. The atomic structure of a truncated Ku70-Ku80 was determined; however, the subunit-specific carboxy-terminal domain of Ku80--essential for binding to DNA-PKcs--was determined only in isolation, and the C-terminal domain of Ku70 was not resolved in its DNA-bound conformation. Both regions are conserved and mediate protein-protein interactions specific to mammals. Here, we reconstruct the three-dimensional structure of the human full-length Ku70-Ku80 dimer at 25 A resolution, alone and in complex with DNA, by using single-particle electron microscopy. We map the C-terminal regions of both subunits, and their conformational changes after DNA and DNA-PKcs binding to define a molecular model of the functions of these domains during DNA repair in the context of full-length Ku70-Ku80 protein.     

Analysis of the kinetic and equilibrium binding of Ku protein to DNA.

The loading of Ku onto a DNA end in a double-strand DNA break is thought to be one of the first steps in the non-homologous DNA end joining (NHEJ) pathway, giving it an essential role in the maintenance of genomic integrity. The binding of Ku to DNA is complicated since DNA can accommodate multiple Ku subunits, which can translocate on the DNA strand. Furthermore, Ku may exhibit cooperativity in the loading process. Therefore, simple one- to-one kinetic models are unable to adequately simulate the process. However, through the use of computer simulation and curve-fitting, we are able to provide a comprehensive mechanistic model and rate constants that closely approximate experimental data for DNA molecules that bind one, two, and three Ku molecules under both kinetic and equilibrium conditions. The model obtains a best fit with Ku having a roughly seven-fold preference to bind to DNA ends rather than internal positions and is consistent with Ku having a strong preference of which face of the protein loads onto the DNA end.     

Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination

DNA double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The DNA cell cycle stage and resection of the DSB ends are two key mechanisms which are believed to push DSB repair to the HR pathway. Here, we show that the NHEJ factor Ku80 associates with DSBs in S phase, when HR is thought to be the preferred repair pathway, and its dynamics/kinetics at DSBs is similar to those observed for Ku80 in non-S phase in mammalian cells. A Ku homolog from Mycobacterium tuberculosis binds to and is retained at DSBs in S phase and was used as a tool to determine if blocking DNA ends affects end resection and HR in mammalian cells. A decrease in DNA end resection, as marked by IR-induced RPA, BrdU, and Rad51 focus formation, and HR are observed when Ku deficient rodent cells are complemented with Mt-Ku. Together, this data suggests that Ku70/80 binds to DSBs in all cell cycle stages and is likely actively displaced from DSB ends to free the DNA ends for DNA end resection and thus HR to occur.     

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-PK-dependent binding of DNA ends to plasmids containing nuclear matrix attachment region DNA sequences: evidence for assembly of a repair complex

We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Purified Ku/DNA-PKcs alone did not produce association of DNA ends with plasmid DNA suggesting that additional factors in the nuclear extract are necessary for this activity. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end binding was observed. Calculation of relative binding activities indicates that DNA end-binding activities to MAR sequences was 7–21-fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV and scaffold attachment factor A preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends suggesting that binding of these proteins to DNA ends is necessary for their association with MAR DNA. The ability of DNA-PKcs/Ku to direct DNA ends to MAR and pUC18 plasmid DNA is a new activity for DNA-PK and may be important for its function in double-strand break repair. A model for DNA repair based on these observations is presented.