Conformational transitions of proteins engaged in DNA double-strand break repair, analysed by tryptophan fluorescence emission and FRET.

We analysed protein-DNA and protein-protein interactions relevant to the repair of DNA DSBs (double-strand breaks) by NHEJ (non-homologous end-joining). Conformational transitions in mammalian DNA ligases III (LigIII) and IV (LigIV), as well as in PARP-1 [poly(ADP-ribose) polymerase-1], were analysed upon binding to double-stranded DNA by changes in tryptophan emission and FRET (Förster resonance energy transfer) from tryptophan to DNA-conjugated Alexa Fluor® 532. For LigIII, two non-equivalent high- and low-affinity DNA-binding sites are detected interacting sequentially with DNA. PARP-1 displays a single high-affinity DNA-binding site and can displace bound DNA fragments from the low-affinity site of LigIII, consistent with its mediator role in LigIII-DNA interactions. For the LX [LigIV-XRCC4 (X-ray cross-complementation group 4)] complex, a single DNA-binding site is detected. Binding of Ku to DNA was accompanied by conformational changes in the protein and intermolecular FRET from dansyl chromophores of the labelled Ku to the Alexa Fluor® chromophores of Alexa Fluor® 532-conjugated DNA. The average distance of 5.7 nm calculated from FRET data is consistent with a location of Ku at the very end of the DNA molecule. Binding of LX to Ku-DNA complexes is associated with conformational changes in Ku, translocating the protein further towards the DNA ends. The protein-protein and protein-DNA interactions detected and analysed generate a framework for the characterization of molecular interactions fundamental to the function of NHEJ pathways in higher eukaryotes.

Metformin Induces a Caspase 3-Unrelated Apoptosis in Human Colorectal Cancer Cell Lines HCT116 and SW620.

Purpose: To study the effects of metformin on the proliferation and growth of human colorectal cancer cell lines HCT116 and SW620. Materials and Methods: The antiproliferative effect of metformin was assayed using an MTS reagent and its ability to inhibit colony formation was demonstrated using a clonogenic assay. Flow cytometry using YO-PRO-1/PI was performed to examine the effects of metformin on apoptosis and cell death of HCT116 and SW620. Caspase 3 activities were measured in caspase-3 activity tests using a caspase-3 activity kit. Furthermore, Western blots were performed with anti-PARP1, anti-caspase 3, and anti-cleaved caspase 3 to confirm whether caspase activation was present or not. Results: Both MTS proliferation assays and clonogenic assays showed that metformin inhibited the proliferation and growth of HCT116 and SW620 cells in a concentration-dependent manner. Flow cytometric analysis identified early apoptosis and metformin-induced cell death in both cell lines. However, caspase 3 activity could not be detected. Cleavage of both PARP1 and pro-caspase 3 was not observed in the Western blot, confirming the absence of caspase 3 activations. Conclusion: This present study suggests a caspase 3-unrelated apoptosis mechanism of metformin-induced cell death in human colorectal cancer cell lines HCT116 and SW620.

Histone H1 functions as a stimulatory factor in backup pathways of NHEJ.

DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ.

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways.

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer.

Marked dependence on growth state of backup pathways of NHEJ.

PURPOSE: Backup pathways of nonhomologous end joining (B-NHEJ) enable cells to repair DNA double-strand breaks (DSBs) when DNA-PK-dependent NHEJ (D-NHEJ) is compromised. Recent evidence implicates growth signaling in the regulation of D-NHEJ. This study was intended to determine whether the ability to repair DSBs by B-NHEJ also depends on growth state. METHODS AND MATERIALS: LIG4(-/-) and wild type (WT) mouse embryo fibroblasts (MEFs) were used. Repair of DSBs was measured by pulsed-field agarose gel electrophoresis. G1 cells were selected by centrifugal elutriation. A plasmid assay was used to measure DNA end-joining activity in whole cell extracts. RESULTS: Wild-type MEFs efficiently repaired DSBs by D-NHEJ in either the exponential or plateau phase of growth. Because of their defect in ligase IV, which compromises D-NHEJ, LIG4(-/-) MEFs showed reduced repair capacity but were slowly able to rejoin a large proportion of DSBs via B-NHEJ. B-NHEJ was markedly reduced in the plateau phase of growth or at high radiation doses. Elutriated G1 cells from exponentially growing or plateau-phase LIG4(-/-) cultures showed a response similar to nonelutriated cells, ruling out that the effect simply reflects redistribution in the cell cycle. An in vitro assay, gauging the activity of B-NHEJ, showed a reduction in DNA end joining during the plateau phase that could be corrected by recombinant DNA ligase IIIalpha. CONCLUSIONS: Suppression of growth signaling markedly compromises DSB repair by B-NHEJ. This effect is associated with a reduction in DNA ligase III mediated DNA end joining.

DNA ligase III as a candidate component of backup pathways of nonhomologous end joining.

Biochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Whereas D-NHEJ depends on the activities of the DNA-dependent protein kinase and DNA ligase IV/XRCC4, components of B-NHEJ have not been identified. Using extract fractionation, we show that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. The same fractionation protocols provide evidence for an additional factor strongly enhancing DNA end joining and shifting the product spectrum from circles to multimers. An in vivo plasmid assay shows that DNA ligase IV-deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNA interference. These in vivo and in vitro observations identify DNA ligase III as a candidate component for B-NHEJ and point to additional factors contributing to NHEJ efficiency.

Plasmid-based assays for DNA end-joining in vitro.

Double-strand breaks (DSBs) disrupt DNA integrity and cause genomic instability and cancer, mutations, or cell death. Among the pathways utilized by cells of higher eukaryotes to repair this lesion, nonhomologous end-joining (NHEJ) is the most dominant. The biochemical characterization of NHEJ has significantly benefited from in vitro plasmid end-joining assays that can complement and extend information obtained from genetic studies. There is evidence that several factors involved in DNA-PK-dependent NHEJ remain to be identified. In addition, under certain circumstances, cells utilize backup pathways of NHEJ that depend on unknown factors to remove DSBs. Characterization of these putative factors will benefit from plasmid-based assays of DNA end-joining. Here, we describe a protocol for in vitro end-joining using plasmid DNA as substrate. The required procedures include: (1) preparation of HeLa-cell nuclear extract; (2) preparation of plasmid substrate DNA; (3) assembly of in vitro DNA repair reactions; and (4) product analysis by gel electrophoresis. The assay is powerful and easy to perform, but one should be aware that it represents an oversimplification, as it does not consider the in vivo organization of DNA into chromatin.

A new chitinase-producer strain Streptomyces glauciniger WICC-A03: isolation and identification as a biocontrol agent for plants phytopathogenic fungi.

This study discusses the isolation and identification of a new Streptomycetes highly active chitinase producer. Fifteen strains were isolated from Malaysian soil samples. The isolate WICC-A03 was found to be the most active chitinase producer. Its antifungal activity was evaluated against many phytopathogens. The identification of WICC-A03 using phenotypic and genotypic methods strongly indicated that strain WICC-A03 belonged to the genus Streptomyces and displayed similarity (91%) with Streptomyces glauciniger. Thus, it was given the suggested name S. glauciniger WICC-A03 with accession number: JX139754. WICC-A03 produces extracellular chitinase in a medium containing 1.5% colloidal chitin in submerged culture on 144 h. The produced enzyme was partially characterised and its molecular weight of 50 kDa was determined by using SDS-PAGE. This study indicates that WICC-A03 is a potential chitinase producer for biocontrol of plant pathogens. Further experiments are being carried out to optimise medium composition and cultivation conditions under lab and bioreactor scale.

Plant proteins, minerals and trace elements of Eurycoma longifolia (Tongkat Ali).

A water extraction method has been used to extract plant proteins from the roots of Eurycoma longifolia harvested from Perak and Pahang, Malaysia. On the basis of the spectroscopic Bradford assay, Tongkat Ali Perak and Pahang contained 0.3868 and 0.9573 mg mL(-1) of crude protein, respectively. The crude proteins were separated by one dimensional 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis into two (49.8 and 5.5 kD) and four (49.8, 24.7, 21.1 and 5.5 kD) protein spots for Tongkat Ali Perak and Pahang, respectively. Isoleucine was present in the highest concentration significantly. Both plant samples showed differences in the mineral and trace element profiles, but the minerals calcium, magnesium and potassium were present in the highest concentration. The highly concerned toxic metals such as arsenic and lead were not detected.

Backup pathways of NHEJ are suppressed by DNA-PK.

In cells of higher eukaryotes double strand breaks (DSBs) induced in the DNA after exposure to ionizing radiation (IR) are rapidly rejoined by a pathway of non-homologous end joining (NHEJ) that requires DNA dependent protein kinase (DNA-PK) and is therefore termed here D-NHEJ. When this pathway is chemically or genetically inactivated, cells still remove the majority of DSBs using an alternative, backup pathway operating independently of the RAD52 epistasis group of genes and with an order of magnitude slower kinetics (B-NHEJ). Here, we investigate the role of DNA-PK in the functional coordination of D-NHEJ and B-NHEJ using as a model end joining by cell extracts of restriction endonuclease linearized plasmid DNA. Although DNA end joining is inhibited by wortmannin, an inhibitor of DNA-PK, the degree of inhibition depends on the ratio between DNA ends and DNA-PK, suggesting that binding of inactive DNA-PK to DNA ends not only blocks processing by D-NHEJ, but also prevents the function of B-NHEJ. Residual end joining under conditions of incomplete inhibition, or in cells lacking DNA-PK, is attributed to the function of B-NHEJ operating on DNA ends free of DNA-PK. Thus, DNA-PK suppresses alternative pathways of end joining by efficiently binding DNA ends and shunting them to D-NHEJ.