Electron microscopy reconstructions of DNA repair complexes

Lesions in DNA compromise the integrity of the genome; their consequences can range from cell malfunction to malignant transformation. DNA damage is repaired by huge multisubunit macromolecular complexes of dynamic composition and conformation. Hence, single-particle electron microscopy has started to contribute significantly to resolving the DNA repair machinery. In many cases, the complexity of the task means that the work requires laborious purification, well-designed strategies for image processing and meticulous labelling of subunits; often, only negative staining is feasible. Recent electron microscopy studies have revealed that the association of DNA-PKcs with Ku70/Ku80 and DNA during non-homologous end joining induces conformational changes that activate the kinase and direct the formation of a synaptic complex. Also, rearrangements of Rad51 filaments and their association with Brca2 were found to regulate homologous recombination.     

Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK

DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.     

Electron microscopy studies of DNA complexes with restriction endonuclease SalGI

The binding properties of the type II restriction endonuclease SalGI to the plasmid DNA pGW 10 has been investigated by electron microscopic studies. Samples were spread by the BAC technique. In the presence of magnesium, SalGI binds as dimers and tetramers to the specific recognition site 5'-G-T-C-G-A-C-3' and with lower rate to the sequence 5'-G-T-C-A-A-C-3', which represents the recognition site of the restriction endonucleases Hind II and Hinc II.     

C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.     

Radiation damage to DNA in DNA-protein complexes

The most aggressive product of water radiolysis, the hydroxyl (OH) radical, is responsible for the indirect effect of ionizing radiations on DNA in solution and aerobic conditions. According to radiolytic footprinting experiments, the resulting strand breaks and base modifications are inhomogeneously distributed along the DNA molecule irradiated free or bound to ligands (polyamines, thiols, proteins). A Monte-Carlo based model of simulation of the reaction of OH radicals with the macromolecules, called RADACK, allows calculating the relative probability of damage of each nucleotide of DNA irradiated alone or in complexes with proteins. RADACK calculations require the knowledge of the three dimensional structure of DNA and its complexes (determined by X-ray crystallography, NMR spectroscopy or molecular modeling). The confrontation of the calculated values with the results of the radiolytic footprinting experiments together with molecular modeling calculations show that: (1) the extent and location of the lesions are strongly dependent on the structure of DNA, which in turns is modulated by the base sequence and by the binding of proteins and (2) the regions in contact with the protein can be protected against the attack by the hydroxyl radicals via masking of the binding site and by scavenging of the radicals.     

Electron microscopy of functional ribosome complexes

Cryoelectron microscopy has made a number of significant contributions to our understanding of the translation process. The method of single-particle reconstruction is particularly well suited for the study of the dynamics of ribosome-ligand interactions. This review follows the events of the functional cycle and discusses the findings in the context provided by the recently published x-ray structures.     

Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

DNA ligase IV is an essential protein that functions in DNA non-homologous end-joining, the major mechanism that rejoins DNA double-strand breaks in mammalian cells. LIG4 syndrome represents a human disorder caused by mutations in DNA ligase IV that lead to impaired but not ablated activity. Thus far, five conserved motifs in DNA ligases have been identified. We previously reported G469E as a mutational change in a LIG4 syndrome patient. G469 does not lie in any of the previously reported motifs. A sequence comparison between DNA ligases led us to identify residues 468-476 of DNA ligase IV as a further conserved motif, designated motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly, however, such mutational changes do impact upon double-stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggest that residues 470-476 function as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.     

Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase

DNA ligase IV (Lig4), x-ray cross-complementation group 4 (XRCC4), and DNA-dependent protein kinase (DNA-PK) are essential mammalian nonhomologous end joining proteins used for V(D)J recombination and DNA repair. Previously a Lig4 peptide was reported to be an in vitro substrate for DNA-PK, but the phosphorylation state of Lig4 protein in vivo is not known. In this study, we report that a full-length Lig4 construct was expressed as a phosphoprotein in the cell. Also the full-length Lig4 protein, in complex with XRCC4, was an in vitro substrate for DNA-PK. Using tandem mass spectrometry, we identified a DNA-PK phosphorylation site at Thr-650 in human Lig4 and a potential second phosphorylation site at Ser-668 or Ser-672. Phosphorylation of Lig4 per se was not required for Lig4 DNA end joining activity. Substitution of these amino acids with alanine, individually or in combination, led to changes in Lig4 protein stability of mouse Lig4. The phosphomimetic mutation S650D returned Lig4 stability to that of the wild-type protein. Furthermore DNA-PK was found to negatively regulate Lig4 protein stability. Our results suggest that Lig4 stability is regulated by multiple factors, including interaction with XRCC4, phosphorylation status, and possibly Lig4 conformation.     

Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule

The DNA ligase IV.XRCC4 complex (LX) functions in DNA non-homologous-end joining, the main pathway for double-strand break repair in mammalian cells. We show that, in contrast to ligation by T4 ligase, the efficiency of LX ligation of double-stranded (ds) ends is critically dependent upon the length of the DNA substrate. The effect is specific for ds ligation, and LX/DNA binding is not influenced by the substrate length. Ku stimulates LX ligation at concentrations resulting in 1-2 Ku molecules bound per substrate, whereas multiply Ku-bound DNA molecules inhibit ds ligation. The combined footprint of DNA with Ku and LX bound is the sum of each individual footprint suggesting that the two complexes are located in tandem at the DNA end. Inhibition of Ku translocation by the presence of cis-platinum adducts on the DNA substrate severely inhibits ligation by LX. Fluorescence resonance energy transfer analysis using fluorophore-labeled Ku and DNA molecules showed that, as expected, Ku makes close contact with the DNA end and that addition of LX can disrupt this close contact. Finally, we show that recruitment of LX by Ku is impaired in an adenylation-defective mutant providing further evidence that LX interacts directly with the DNA end, possibly via the 5'-phosphate as shown for prokaryotic ligases. Taken together, our results suggest that, when LX binds to a Ku-bound DNA molecule, it causes inward translocation of Ku and that freedom to move inward on the DNA is essential to Ku stimulation of LX activity.     

Electron microscopy of DNA.helicase-I complexes in the act of strand separation

Electron microscopy was used to characterize the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase I. Linear DNA with 5'-protruding strands as well as single-stranded gaps was incubated, under unwinding assay conditions, with the helicase. E. coli single-stranded-DNA-binding protein (SSB) was added to order the denatured DNA. Up to 70% of the sites of SSB-complexed DNA were observed as forks. The position of the strand-separating enzyme was indicated by a gap in the complex between fork and SSB on that arm which initially provided the binding site. The complex between DNA and helicase varied in length although in all cases it was long enough to comprise several helicase I molecules. A mutant helicase I (helicase I del29) which, unlike the wild-type enzyme, fails to show cooperative DNA-binding behaviour was found to prevent an abnormally short stretch of DNA near the fork from binding SSB. Apparently, one or very few helicase molecules would be sufficient for the opening of a DNA duplex although, typically, the fork is shifted by a tract of helicase I molecules. SSB displaces helicase I from single-stranded DNA but fails to do so from a fork or a single-strand/double-strand junction. The difference is consistent with the observation that SSB does not inhibit the unwinding reaction despite its rapid association with the separated strands. Helicase I unwinds in the 5'-3' direction of the bound strand. Observations so far indicate that the enzyme exploits the single strand at the initial DNA-binding site for orienting its action, and not the complementary, completely base-paired strand.     

Human DNA ligase IV and the ligase IV/XRCC4 complex: analysis of nick ligation fidelity

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.     

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.     

DNA-protein complexes of the nuclear matrix: visualization and partial characterization of the protein component

Complexes composed of DNA attached to the nuclear matrix and of proteins most tightly bound to DNA are visualized as globular particles 25-35 nm in diameter. Their morphology depends greatly on the isolation conditions: a Cs salts/urea combination permits the isolation while CsCl/sarcosyl destroys the particles. The preparation is shown to have the same protein content regardless of the treatment employed. The proteins of the complex are resistant to SDS and pronase treatment and to phenol/chloroform extraction while being associated with DNA.     

Immunoprecipitation of DNA-protein complexes cross-linked by cis-diamminedichloroplatinum

For the study of in vitro and in vivo DNA-protein interactions, cross-linking reactions driven by UV or formaldehyde have been frequently used, followed by standard protocols of immunoprecipitation and analysis of the DNA isolated from the complexes. Here we present a basically modified method to analyze the DNA-protein cross-linked complexes obtained by an alternative cross-linking reagent. The innovations presented here include cross-linking by cis-diamminedichloroplatinum II, a fast method to isolate DNA-protein complexes using gel-filtration chromatography, and a modified procedure to obtain specific immunocomplexes that can be analyzed either for DNA or for protein content. The application of this method to two nuclear proteins from chicken liver nuclei is described.