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PubMed2006

Insights into the mechanism and catalysis of the native chemical ligation reaction.

Native chemical ligation of unprotected peptide segments involves reaction between a peptide-alpha-thioester and a cysteine-peptide, to yield a product with a native amide bond at the ligation site. Peptide-alpha-thioalkyl esters are commonly used because of their ease of preparation. These thioalkyl esters are rather unreactive so the ligation reaction is catalyzed by in situ transthioesterification with thiol additives. The most common thiol catalysts used to date have been either a mixture of thiophenol/benzyl mercaptan, or the alkanethiol MESNA. Despite the use of these thiol catalysts, ligation reactions typically take 24-48 h. To gain insight into the mechanism of native chemical ligaton and in order to find a better catalyst, we investigated the use of a number of thiol compounds. Substituted thiophenols with pK(a) > 6 were found to best combine the ability to exchange rapidly and completely with thioalkyl esters, and to then act as effective leaving groups in reaction of the peptide-thioester with the thiol side chain of a cysteine-peptide. A highly effective and practical catalyst was (4-carboxylmethyl)thiophenol ('MPAA'), a nonmalodorous, water-soluble thiol. Use of MPAA gave an order of magnitude faster reaction in model studies of native chemical ligation and in the synthesis of a small protein, turkey ovomucoid third domain (OMTKY3). MPAA should find broad use in native chemical ligation and in the total synthesis of proteins.

Catalysis↗
PubMed1996

Nonenzymatic, template-directed ligation of oligoribonucleotides is highly regioselective for the formation of 3'-5' phosphodiester bonds.

We have found that nonenzymatic, template-directed ligation reactions of oligoribonucleotides display high selectivity for the formation of 3'-5' rather than 2'-5' phosphodiester bonds. Formation of the 3'-5'-linked product is favored regardless of the metal ion catalyst or the leaving group, and for several different ligation junction sequences. The degree of selectivity depends on the leaving group: the ratio of 3'-5'- to 2'-5'-linked products was 10-15:1 when the 5'-phosphate was activated as the imidazolide, and 60-80:1 when the 5'-phosphate was activated by the formation of a 5'-triphosphate. Comparison of oligonucleotide ligation reactions with previously characterized single nucleotide primer extension reactions suggests that the strong preference for 3'-5'-linkages in oligonucleotide ligation is primarily due to occurence of ligation within the context of an extended Watston-Crick duplex. The ability of RNA to correctly self-assemble by template-directed ligation is an intrinsic consequence of its chemical structure and need not be imposed by an external catalyst (i.e., an enzyme polymerase); RNA therefore provides a reasonable structural basis for a self-replicating system in a prebiological world.

Base Sequence↗
PubMed1998

Formation of oligonucleotide-PNA-chimeras by template-directed ligation.

DNA sequences have previously been reported to act as templates for the synthesis of PNA, and vice versa. A continuous evolutionary transition from an informational replicating system based on one polymer to a system based on the other would be facilitated if it were possible to form chimeras, that is molecules that contain monomers of both types. Here we show that ligation to form chimeras proceeds efficiently both on PNA and on DNA templates. The efficiency of ligation is primarily determined by the number of backbone bonds at the ligation site and the relative orientation of template and substrate strands. The most efficient reactions result in the formation of chimeras with ligation junctions resembling the structures of the backbones of PNA and DNA and with antiparallel alignment of both components of the chimera with the template, that is, ligations involving formation of 3'-phosphoramidate and 5'-ester bonds. However, double helices involving PNA are stable both with antiparallel and parallel orientation of the two strands. Ligation on PNA but not on DNA templates is, therefore, sometimes possible on templates with reversed orientation. The relevance of these findings to discussions of possible transitions between genetic systems is discussed.

DNA↗
PubMed2001

Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells.

Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP-70 kinase, or TCR/CD3 gamma-chain showed that the cells undergo apoptosis after Fas ligation. Anisomycin exposure induced apoptosis in the src p56(lck)-deficient cell line but not in the two other mutant cell lines. Simultaneous cross-linking of MHC-I and Fas ligation inhibited apoptosis in the ZAP-70 kinase and the TCR/CD3 gamma-chain mutants, but did not protect the src p56(lck)-deficient cells. Similarly, MHC-I ligation did not protect anisomycin-treated src p56(lck)-deficient cells against apoptosis. These data suggest that MHC-I-induced inhibition of apoptosis depends on intact src p56(lck) activity, but not on major secondary messenger molecules associated with TCR signaling. Overall the results support the idea that signal transduction by MHC-I molecules is involved in homeostatic processes of importance for T-cell survival and death.

Anisomycin↗
PubMed2004

Evaluation of the frictional resistance of conventional and self-ligating bracket designs using standardized archwires and dental typodonts.

The frictional behavior of four conventional and four self-ligating brackets were simulated using a mechanical testing machine. Analyses of the two-bracket types were completed by drawing samples of three standardized archwires through quadrants of typodont models in the dry/wet states. Pretreatment typodonts of an oral cavity featured progressively malocclused quadrants. As nominal dimensions of the archwires were increased, the drawing forces of all brackets increased at different rates. When coupled with a small wire, the self-ligating brackets performed better than the conventional brackets. For the 0.014-inch wires in the upper right quadrant, the maximum drawing forces averaged 125 and 810 cN for self-ligating and conventional brackets, respectively. When coupled with larger wires, various designs interchangeably displayed superior performance. For the 0.019- x 0.025-inch wires in the upper left quadrant, the maximum drawing forces averaged 1635 and 2080 cN for self-ligating and conventional brackets, respectively. As the malocclusion increased, the drawing forces increased. For example, in the least malocclused quadrant and with the smallest wire, maximum drawing forces for self-ligating and conventional brackets averaged 80 and 810 cN, respectively, whereas in the most malocclused quadrant tested with the same wire size, maximum drawing forces for self-ligating and conventional brackets averaged 870 and 1345 cN, respectively. For maximum values between the dry and wet states, significant differences between ambient states existed only for the In-Ovation brackets in the lower left quadrant. These test outcomes illustrated how bracket design, wire size, malocclusion, and ambient state influenced drawing forces.

Dental Alloys↗
PubMed2005

Archwire ligation techniques, microbial colonization, and periodontal status in orthodontically treated patients.

Fixed or removable orthodontic appliances impede the maintenance of oral hygiene and result in plaque accumulation. Plaque retention surrounding orthodontic appliances leads to enamel demineralization caused by organic acids produced by bacteria in the dental plaque. Many studies have evaluated the effects of fixed orthodontic appliances on microbial flora and periodontal status, but only a few have evaluated the method of ligation as an additional factor. The aim of this study was to determine the changes in microbial flora and periodontal status after orthodontic bonding and to determine whether two different archwire ligation techniques affect these changes. A total of 21 orthodontic patients scheduled for fixed orthodontic treatment were selected for this split-mouth study. Two commonly used auxiliaries (elastomeric rings and ligature wires) for tying archwires were tested. Microbial and periodontal records were obtained before bonding (T0), one week later (T1), and five weeks after bonding (T2). Paired t-test and Wilcoxon signed rank test were used to compare the groups statistically. Although, teeth ligated with elastomeric rings exhibited slightly greater numbers of microorganisms than teeth ligated with steel ligature wires, the differences were not statistically significant and could be ignored. The two archwire ligation techniques showed no significant differences in the gingival index, bonded bracket plaque index, or pocket depths of the bonded teeth. However, teeth ligated with elastomeric rings were more prone to bleeding. Therefore, elastomeric ring use is not recommended in patients with poor oral hygiene.

Adolescent↗
PubMed1999

Vascular surgical society of great britain and ireland: outcome after ligation of infected false femoral aneurysms in intravenous drug abusers

BACKGROUND: Infected false femoral aneurysm (IFFA) is a life-threatening complication of intravenous drug abuse and presents a difficult management problem for the vascular surgeon. Controversy exists regarding the best management. The choice lies between ligation and excision with immediate revascularization, and ligation and excision with observation; reconstruction is reserved for critical ischaemia. METHODS: After disappointing results with the former method it was decided to perform ligation and excision with observation as the initial treatment of IFFA. A 9-year experience of 28 patients treated at this hospital is reviewed. RESULTS: In 26 cases of primary ligation and excision of an IFFA there were no amputations and patients described claudication only in follow-up. In two cases of a second IFFA in the same limb, repeat ligation and excision resulted in one viable limb with claudication only and one above-knee amputation for non-viability. At 9-year follow-up (80 per cent complete), over 90 per cent of the patients were still drug abusers and therefore not suitable for revascularization. There were two deaths, both of which were drug related. CONCLUSION: Ligation and excision of an IFFA is simple, effective and safe, and is the most appropriate method of dealing with these challenging patients.

Journal Article↗
PubMed2001

Upregulation of keratinocyte growth factor in the tracheal ligation lamb model of congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) carries a high mortality rate of 60% because of associated anomalies, pulmonary hypoplasia, pulmonary hypertension, and type II cell dysfunction. Prenatal tracheal ligation has been shown to improve lung growth in experimental models. This could be caused by a direct effect of increased endothoracic pressure in utero, secondary to the induction of specific growth factors, or both. Keratinocyte growth factor (KGF) is involved in normal lung organogenesis and is a potent mitogen of alveolar type II cells. The authors have therefore investigated the protein and mRNA levels of keratinocyte growth factor in the lung tissue of control, CDH, and CDH tracheal ligation lambs. METHODS: Eight lambs had left-sided diaphragmatic hernias surgically created at 80 days' gestation. Tracheal ligation was performed at 110 days in 4 lambs, and they were delivered by cesarean section at 140 to 145 days. Twin littermates served as controls. The lungs were dissected en bloc and snap frozen. KGF protein levels were determined by ELISA. Total RNA was isolated, and a RNase protection assay was performed using an ovine cDNA probe for KGF, and a human cDNA probe for GAPDH (house keeping control). Densitometric analysis was used to quantify the relative amounts of mRNA in each sample. RESULTS: There was a significant decrease in the KGF protein levels of the CDH samples (110 v 73.2 pg/mg protein, P =.02). This decrease was mirrored by a significant fall in the level of mRNA expression for KGF (0.694 v 0.235, P = .02). Tracheal ligation normalized the KGF protein levels (96.1 pg/mg protein). This elevation of KGF protein was accompanied by an upregulation of KGF gene expression to control levels (0.56). CONCLUSIONS: Tracheal ligation clearly is accompanied by an upregulation of keratinocyte growth factor protein and gene expression. It is not yet clear whether keratinocyte growth factor is solely responsible for the growth observed in these tracheal ligation preparations. Further growth factor blocking experiments are required.

Animals↗
PubMedDate not supplied

Sodium nitroprusside-induced deliberate hypotension to facilitate patent ductus arteriosus ligation in dogs.

OBJECTIVE: To report the use of sodium nitroprusside to induce deliberate hypotension to reduce hemorrhage and facilitate surgical ligation of complicated patent ductus arteriosus (PDA) in dogs. STUDY DESIGN: Retrospective study. ANIMALS: Six dogs. METHODS: Hemorrhage occurred during surgical ligation of PDA in 5 dogs. Surgical dissection and isolation of the PDA were very difficult in a sixth dog that was considered to be at increased risk for ductus rupture. Sodium nitroprusside (5 to 25 microg/kg/min intravenously) was administered to these 6 dogs to induce hypotension to reduce blood loss and facilitate surgical ligation of the PDA. RESULTS: Sodium nitroprusside infusion reduced blood pressure (mean arterial pressure, 45 to 60 mm Hg) within 5 to 10 minutes and hemorrhage from the PDA, facilitating its surgical ligation. Recovery from surgery and anesthesia was uneventful in all 6 dogs. CONCLUSIONS: Sodium nitroprusside infusion can be used to induce deliberate hypotension in dogs to facilitate surgical ligation of PDA. CLINICAL RELEVANCE: Sodium nitroprusside infusion produces hypotension within 5 to 10 minutes and is easy to control, rapidly abates, and should help to facilitate surgical ligation of PDA.

Animals↗
PubMed2000

Tubal ligation and risk of breast cancer.

Although it has been demonstrated in previous studies that tubal ligation can have widespread effects on ovarian function, including a decrease in the risk of subsequent ovarian cancer, few studies have evaluated effects on breast cancer risk. In a population-based case-control study of breast cancer among women 20-54 years of age conducted in three geographic areas, previous tubal ligations were reported by 25.3% of the 2173 cases and 25.8% of the 1990 controls. Initially it appeared that tubal ligations might impart a slight reduction in risk, particularly among women undergoing the procedure at young ages (<25 years). However, women were more likely to have had the procedure if they were black, less educated, young when they bore their first child, or multiparous. After accounting for these factors, tubal ligations were unrelated to breast cancer risk (relative risk (RR) = 1.09, 95% confidence interval (CI) 0.9-1.3), with no variation in risk by age at, interval since, or calendar year of the procedure. The relationship of tubal ligations to risk did not vary according to the presence of a number of other risk factors, including menopausal status or screening history. Furthermore, effects of tubal ligation were similar for all stages at breast cancer diagnosis. Further studies would be worthwhile given the biologic plausibility of an association. However, future investigations should include information on type of procedure performed (since this may relate to biologic effects) as well as other breast cancer risk factors.

Adult↗
PubMed2001

Immediate reorganization of the rat somatosensory thalamus after partial ligation of sciatic nerve.

Nerve injury can result in neuropathic pain, which persists after the injury and may occur after healing is completed. The long-term central reorganization associated with neuropathic pain has been previously studied in animal models. The immediate effects of nerve injury on central representation, however, are poorly understood. We examined the population response properties of closely neighboring neurons located in the hindlimb representation area of the somatosensory thalamus. Changes in the neuronal population properties were characterized before, during, and after (up to 6 hours) partial ligation of the sciatic nerve in the rat. Changes in these properties were observed within minutes after nerve injury. There were changes in neuronal class and receptive field size, emergence of new receptive fields, receptive fields observed before ligation disappeared temporarily after ligation, and changes in number of spikes evoked by the same stimulus. The rates of these changes in central representation were essentially zero before ligation, maximal within minutes after ligation, and decreased to a steady sustained rate of change within 1 to 2 hours. The incidence of functional connectivity, as measured by cross-correlations, remained unchanged. However, the strength of functional connectivity increased after ligation. The results show immediate reorganization of lateral thalamic networks with peripheral nerve damage. When the population response is considered as the underlying code, this reorganization does not reflect the behavioral manifestations of hyperalgesia and allodynia, even though some of the individual neuronal responses do reflect properties consistent with the hyperalgesia and allodynia reported within the same time frame after nerve injury in the rat.

Journal Article↗
PubMed2003

CTCF functions as a critical regulator of cell-cycle arrest and death after ligation of the B cell receptor on immature B cells.

The WEHI 231 B cell lymphoma is used as a model of self-tolerance by clonal deletion because B cell receptor (BCR) ligation results in apoptosis. Two critical events precede cell death: an early rise and fall in expression of MYC and cell-cycle arrest associated with enhanced expression of p21, p27, and p53. CTCF is a transcription factor identified as a repressor of MYC recently shown to cause cell growth inhibition. The present studies demonstrate that BCR ligation of WEHI 231 as well as of normal immature B cells greatly increased expression of CTCF in association with down-regulation of MYC followed by growth arrest and cell death. Conditional expression of CTCF in WEHI 231 mimicked BCR ligation with activated cells showing repressed expression of MYC, enhanced expression of p27, p21, p53, and p19(ARF), and inhibition of cell growth and induction of apoptosis. In keeping with a central role for CTCF in control of B cell death, conditional expression of a CTCF antisense construct in WEHI 231 resulted in inhibition of p27, p21, p53, and p19(ARF) in association with enhanced expression of MYC. Activation of the endogenous CTCF locus by BCR ligation was also mimicked by three other routes to apoptotic death in WEHI 231: inhibition of the phosphoinositide 3-kinase or mTORFRAP signaling cascades and treatment with transforming growth factor (TGF)-beta. Rapid activation of CTCF by BCR ligation or treatment with TGF-beta was suppressed by ligation of CD40. These results demonstrate that CTCF is a common determinant to different pathways of death signaling in immature B cells.

Apoptosis↗
PubMed2001

Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary.

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet(7)]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the alpha-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet(7)]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by approximately 45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.

Amino Acid Sequence↗
PubMed1997

A critical role of Lyn and Fyn for B cell responses to CD38 ligation and interleukin 5.

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induced proliferation, interleukin 5 (IL-5) receptor alpha chain expression, and tyrosine phosphorylation of Bruton tyrosine kinase (Btk) from wild-type, but not from X chromosome-linked, immunodeficient mice. B cells from fyn-deficient (Fyn-/-) and lyn-deficient (Lyn-/-) mice showed an impaired response to mAb CS/2 for proliferation and IL-5 receptor alpha chain expression, and B cells from fyn/lyn double-deficient (Fyn/Lyn-/-) mice did not respond at all to mAb CS/2. The Btk activation by CD38 ligation was observed in B cells from Fyn-/- mice, and it was severely impaired in B cells from Lyn-/- and Fyn/Lyn-/- mice. CD38 expression on B cells from three mutant strains was comparable to that on control B cells. We infer from these results that both Fyn and Lyn are required and that their signals are synergistic for B cell triggering after CD38 ligation. Lyn is upstream of Btk activation in the CD38 signaling. Stimulation of B cells with IL-5 together with CD38 ligation induces not only IgM but also IgG1 secretion. Analysis of the synergistic effects of IL-5 and CD38 ligation on IgG1 secretion revealed the impaired IgG1 secretion of B cells from Lyn-/- and Fyn/Lyn-/- mice. These data imply that Lyn is involved in B cell triggering by CD38 ligation plus IL-5 for isotype switching.

ADP-ribosyl Cyclase↗
PubMed1999

Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

Amino Acid Sequence↗
PubMed1995

Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fc gamma RII or Fc gamma RIIIB ligation in human polymorphonuclear leukocytes. Implications for immune complex activation of the respiratory burst.

Two tyrosine kinase-dependent pathways exist for activation of the respiratory burst by polymorphonuclear leukocyte (PMN) immunoglobulin G Fc receptors. Direct ligation of Fc gamma RII activates the respiratory burst, but ligation of the glycan phosphoinositol-linked Fc gamma RIIIB does not. Instead, this receptor and the integrin complement receptor CR3 synergize in activation of the respiratory burst (Zhou, M.-J., and Brown, E. J. (1994) J. Cell Biol. 125, 1407-1416). Here we show that direct ligation of Fc gamma RII leads to activation and Triton X-100 insolubility of the Src family kinase Fgr, without effect on the related myeloid Src family member Hck. In contrast, adhesion of PMN via Fc gamma RIIIB leads to activation and Triton X-100 insolubility of Hck but not Fgr. The exclusive association of Fc gamma RIIIB with Hck activation and Triton insolubility is not solely a result of its glycan phosphoinositol anchor, since decay accelerating factor (CD55), another prominent glycan phosphoinositol-anchored PMN protein, is associated with Fgr insolubility to a greater extent than Hck. Ligation of decay accelerating factor, with or without coligation of CR3, does not activate the PMN respiratory burst. Coligation of Fc gamma RIIIB with Fc gamma RII overcomes the pertussis toxin inhibition of H2O2 production in response to direct ligation of Fc gamma RII. These data support the hypothesis that activation of Hck upon Fc gamma RIIIB ligation has a role in generation of the synergistic respiratory burst.

Antigen-Antibody Complex↗
PubMed2003

Biophysical and structural analysis of a novel heme B iron ligation in the flavocytochrome cellobiose dehydrogenase.

The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 A resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N delta 1 and N epsilon 2 nitrogen atoms, rather than the typical N epsilon 2/N epsilon 2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N delta 1/N epsilon 2-coordinated protoporphyrin IX iron.

Blotting, Western↗
PubMed2003

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.

Antigens, Nuclear↗