Improvements of DNA sequencing gels

We describe three simple modifications of DNA sequencing gels which all result in improved oligonucleotide resolution as visualized by autoradiography. First, it was possible to reduce the thickness of the gel to 0.2 mm by using new gel molding techniques. Second, the gel could be dried without any distortions of its dimensions by prior binding of the gel to the surface of the glass plate. Third, a uniform high temperature was obtained in all parts of the gel during electrophoresis by replacing one of the glass plates with an inexpensive thermostating plate with circulating water. The use of this heating plate resulted in a straight band pattern all over the gel and also in the resolution of such bands which were not resolved in other electrophoresis systems.     

Biosynthesis of mammalian DNA ligase

A monospecific antibody against calf thymus DNA ligase composed of a single polypeptide with Mr = 130,000 cross-reacts with rodent and calf thymus DNA ligases. The antibody precipitates a single Mr = 200,000 polypeptide from detergent lysates of [3H] leucine-labeled mouse Ehrlich tumor cells and calf thymocytes. Pulse-chase experiments show that the Mr = 200,000 polypeptide in Ehrlich tumor cells has a half-life of about 0.5 h. In addition to the Mr = 200,000 polypeptide, a Mr = 130,000 polypeptide is detected in the partially purified enzyme preparations from radiolabeled Ehrlich tumor cells. These results suggest that DNA ligase is synthesized in mammalian cells as a Mr = 200,000 polypeptide and that the Mr = 200,000 polypeptide is degraded to a Mr = 130,000 polypeptide by a limited proteolysis in vitro.     

DNA sequencing separations in capillary gels on a modified commercial DNA sequencing instrument

DNA sequencing separations of standard DNA fragments of known sequence have been achieved in small diameter capillary gels electrophoresed and analyzed in parallel in a modified commercial DNA sequencer instrument. DNA sequencing in terms of base-calling accuracy is comparable to conventional slab gels; however, the separations in the capillary were performed somewhat faster and required less sample than those in the slab gel. Advantages of this approach vs. separations on conventional slab gels are discussed.     

DNA ligase I mediates essential functions in mammalian cells.

DNA replication, repair, and recombination are essential processes in mammalian cells. Hence, the application of gene targeting to the study of these DNA metabolic pathways requires the creation of nonnull mutations. We have developed a method for introducing partially defective mutants in murine embryonic stem cells that circumvents the problem of cellular lethality of targeted mutations at essential loci. Using this approach, we have determined that mammalian DNA ligase I is essential for cell viability. Thus, DNA ligases II and III are not redundant with DNA ligase I for the function(s) associated with cell proliferation. Partial complementation of the lethal DNA ligase I null mutation allowed the creation of deficient embryonic stem cell lines. We found that a wild-type DNA ligase I cDNA, as well as a variant DNA ligase I cDNA, was able to rescue the lethality of the homozygous null mutation, whereas an N-terminal deletion mutant consisting of the minimal DNA ligase I catalytic domain was not. This observation demonstrates that sequences outside the DNA ligase I catalytic domain are essential for DNA ligase I function in vivo.     

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.     

DNA ligase activity in UV-irradiated monkey kidney cells.

The DNA ligase activity of monkey kidney CV-1 cells has been measured at different stages of culture growth and after different time intervals following ultraviolet irradiation. Results indicate that: - The level of enzyme activity is about twice higher in non synchronous, rapidly dividing cells than in confluent cultures. - UV-irradiation of cells induces a "de novo" synthesis of DNA ligase. - This induction is dose dependent in its extent and kinetics, and may lead to a DNA ligase level in UV-irradiated stationary cultures of the same order as observed in unirradiated exponentially growing cells. - This induction seems to be independent of semiconservative DNA synthesis since it is not affected by fluorodeoxyuridine.     

Activation of mammalian DNA ligase I through phosphorylation by casein kinase II.

Mammalian DNA ligase I has been shown to be a phosphoprotein. Dephosphorylation of purified DNA ligase I causes inactivation, an effect dependent on the presence of the N-terminal region of the protein. Expression of full-length human DNA ligase I in Escherichia coli yielded soluble but catalytically inactive enzyme whereas an N-terminally truncated form expressed activity. Incubation of the full-length preparation from E. coli with purified casein kinase II (CKII) resulted in phosphorylation of the N-terminal region and was accompanied by activation of the DNA ligase. Of a variety of purified protein kinases tested, only CKII stimulated the activity of calf thymus DNA ligase I. Tryptic phosphopeptide analysis of DNA ligase I revealed that CKII specifically phosphorylated a major peptide also apparently phosphorylated in cells, implying that CKII is a protein kinase acting on DNA ligase I in the cell nucleus. These data suggest that DNA ligase I is negatively regulated by its N-terminal region and that this inhibition can be relieved by post-translational modification.     

A novel DNA joining activity catalyzed by T4 DNA ligase.

The use of T4 and E. coli DNA ligases in genetic engineering technology is usually associated with nick-closing activity in double stranded DNA or ligation of 'sticky-ends' to produce recombinant DNA molecules. We describe in this communication the ability of T4 DNA ligase to catalyze intramolecular loop formation between annealed oligodeoxyribonucleotides wherein Watson-Crick base pairing is absent on one side of the ligation site. Enzyme concentration, loop size, substrate specificity, and base composition were explored in an effort to maximize yield. Amounts of T4 DNA ligase in large molar excess to DNA template and ligated product are necessary to achieve high yields.     

Vaccinia virus encodes a polypeptide with DNA ligase activity.

Vaccinia virus gene SalF 15R potentially encodes a polypeptide of 63 kD which shares 30% amino acid identity with S. pombe and S. cerevisiae DNA ligases. DNA ligase proteins can be identified by incubation with alpha-(32P)ATP, resulting in the formation of a covalent DNA ligase-AMP adduct, an intermediate in the enzyme reaction. A novel radio-labelled polypeptide of approximately 61 kD appears in extracts from vaccinia virus infected cells after incubation with alpha-(32P)ATP. This protein is present throughout infection and is a DNA ligase as the radioactivity is discharged in the presence of either DNA substrate or pyrophosphate. DNA ligase assays show an increase in enzyme activity in cell extracts after vaccinia virus infection. A rabbit antiserum, raised against a bacterial fusion protein of beta-galactosidase and a portion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts of vaccinia virus-infected cells. This antiserum also immune-precipitates the novel DNA ligase-AMP adduct, thus proving that the observed DNA ligase is encoded by SalF 15R.     

Rejoining of DNA strand breaks by T4 DNA ligase in mammalian cells

We have tested the ability of T4 DNA ligase to rejoin radiation-induced DNA strand breaks in living hamster cells (CHO-K1, EM9, xrs-5). T4 DNA ligase was introduced into cells by electroporation prior to x-irradiation. Single- and double-strand breaks were measured by the alkaline comet assay technique, and double-strand breaks (DSBs) were evaluated by the pulsed-field gel electrophoresis method. In the comet assay, the three cell lines showed reduced tail moments following pretreatment with T4 DNA ligase, both directly after irradiation and after repair incubation for 4 h. Similarly, the results obtained from pulsed-field gel electrophoresis showed reduced DSB frequencies after pretreatment with T4 DNA ligase. We conclude that exogeneous T4 ligase contributes to rejoining of radiation-induced strand breaks.     

DNA ligase activity in carcinogen-treated human fibroblasts

In an enzymological approach to study DNA repair mechanisms induced by carcinogen-treatment of mammalian cells, we have investigated how DNA ligase activity is affected by the treatment with several compounds producing different DNA lesions. Stationary cultures of human fibroblasts were exposed to various doses of carcinogens (UV-light at 254 nm, N-acetoxy-acetyl-aminofluorene, ethyl-methane sulfonate, N-methylnitro-nitrosoguanidine, mitomycin C and 4-nitroquinoline-N-oxide) at different time-intervals before preparing crude cellular extracts and assaying for ligase activity. Results have shown that: 1. UV-irradiation, AAAF, 4NQO or MMC treatment of cells induces a two-fold increase in the ligase activity compared to control cells within 48 hours following the treatment. 2. A partial purification of the enzyme from these cellular crude extracts by sedimentation through sucrose gradients has shown: a. DNA ligase activity from control cells presents a profile composed of two distinct peaks sedimenting respectively at about 4S and 7S; b. the carcinogen treatment of either repair-proficient human fibroblasts or repair-deficient xeroderma pigmentosum cells (complementation group A) seems to induce a specific increase of the 4S-form of DNA ligase.     

Using a neural network for lane-tracking of DNA sequencing slab gels

High quality lane-tracking of gel images is the first task, and thus a prerequisite, for successful trace processing and base-calling of DNA sequencing slab gels. In most approaches, it is based on statistical calculations, for instance variance and co-variance analysis between neighboring pixel columns in the image. On the basis of these statistical calculations, Kohonen's self-organization neural network model was introduced. We have found that, using several well-structured input data, Kohonen's self-organization neural network model can be trained to fulfill our task of lane-tracking. Furthermore, the quality of lane-tracking could be improved compared to algorithmic approaches.     

Deficiency of DNA ligase activity in Fanconi's anemia

Summary A significant decrease in DNA ligase activity was observed in lymphocytes and fibroblasts of a patient with Fanconi's anemia (FA). This decrease is related to the observed DNA repair deficiency indicated by the delayed closing of repair DNA strands following UV irradiation. Other steps of DNA repair were analyzed in the FA fibroblasts, including endonucleolytic incision of DNA, repair DNA synthesis, and exonucleolytic removal of the photoproducts. No differences were found against control cells. The action of DNA ligase is delayed during replication in the FA cells, as seen by an accumulation of replicative intermediates.