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DNA double-strand breaks (DSB) may arise either spontaneously during cellular processes or as a result of exposure to DNA-damaging agents such as ionizing radiation, or radiomimetic agents such as restriction endonucleases or bleomycin. It is widely accepted that nonrepaired or misrepaired DSB are the main lesions leading to the production of chromosomal aberrations, mutagenesis, oncogenic transformation, and cell killing. Studies focusing on this relationship, as well as the possible modulation of DNA repair mechanisms, are currently of major interest. A wide variety of test systems are available to study DNA damage. In the last few years, single-cell gel electrophoresis, commonly known as "comet assay," has been considered a rapid, sensitive, and visual method for quantifying DNA strand breaks and alkali-labile damage in individual cells. In this study, making use of the comet assay, we tried to find out if under conditions that maintain chromatin structure the DNA ligase from T4 phage is able to facilitate the rejoining of strand breaks with different end structures, induced by the restriction endonuclease MspI or bleomycin in living human lymphocytes in a nonproliferating state. T4 DNA ligase, as well as the restriction endonuclease or bleomycin, were introduced together by electroporation into human lymphocytes. Our results support the idea that it is possible to modulate the DSB-rejoining of different DNA strand-breaking agents by exogenous T4 DNA ligase.
Environ Mol Mutagen 1998
PMID:Protection provided by exogenous DNA ligase in G0 human lymphocytes treated with restriction enzyme MspI or bleomycin as shown by the comet assay. 988 8

Endonuclease III from Escherichia coli is the prototype of a ubiquitous DNA repair enzyme essential for the removal of oxidized pyrimidine base damage. The yeast genome project has revealed the presence of two genes in Saccharomyces cerevisiae, NTG1 and NTG2, encoding proteins with similarity to endonuclease III. Both contain the highly conserved helix-hairpin-helix motif, whereas only one (Ntg2) harbors the characteristic iron-sulfur cluster of the endonuclease III family. We have characterized these gene functions by mutant and enzyme analysis as well as by gene expression and intracellular localization studies. Targeted gene disruption of NTG1 and NTG2 produced mutants with greatly increased spontaneous and hydrogen peroxide-induced mutation frequency relative to the wild type, and the mutation response was further increased in the double mutant. Both enzymes were found to remove thymine glycol and 2, 6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (faPy) residues from DNA with high efficiency. However, on UV-irradiated DNA, saturating concentrations of Ntg2 removed only half of the cytosine photoproducts released by Ntg1. Conversely, 5-hydroxycytosine was removed efficiently only by Ntg2. The enzymes appear to have different reaction modes, as judged from much higher affinity of Ntg2 for damaged DNA and more efficient borhydride trapping of Ntg1 to abasic sites in DNA despite limited DNA binding. Northern blot and promoter fusion analysis showed that NTG1 is inducible by cell exposure to DNA-damaging agents, whereas NTG2 is constitutively expressed. Ntg2 appears to be a nuclear enzyme, whereas Ntg1 was sorted both to the nucleus and to the mitochondria. We conclude that functions of both NTG1 and NTG2 are important for removal of oxidative DNA damage in yeast.
Mol Cell Biol 1999 May
PMID:The Saccharomyces cerevisiae homologues of endonuclease III from Escherichia coli, Ntg1 and Ntg2, are both required for efficient repair of spontaneous and induced oxidative DNA damage in yeast. 1020 1

We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzyme. First, the DNA ligase III cDNA contains an in-frame ATG located upstream from the putative translation initiation start site. The DNA sequence between these two ATG sites encodes an amphipathic helix similar to previously identified mitochondrial targeting peptides. Second, recombinant green fluorescent protein harboring this sequence at its amino terminus was efficiently targeted to the mitochondria of Cos-1 monkey kidney cells. In contrast, native green fluorescent protein distributed to the cytosol. Third, a series of hemagglutinin-DNA ligase III minigene constructs were introduced into Cos-1 cells, and immunocytochemistry was used to determine subcellular localization of the epitope-tagged DNA ligase III protein. These experiments revealed that inactivation of the upstream ATG resulted in nuclear accumulation of the DNA ligase III protein, whereas inactivation of the downstream ATG abolished nuclear localization and led to accumulation within the mitochondrial compartment. Fourth, mitochondrial protein extracts prepared from human cells overexpressing antisense DNA ligase III mRNA possessed substantially less DNA ligase activity than did mitochondrial extracts prepared from control cells. DNA end-joining activity was also substantially reduced in extracts prepared from antisense mRNA-expressing cells. From these results, we conclude that the human DNA ligase III gene encodes both nuclear and mitochondrial enzymes. DNA ligase plays a central role in DNA replication, recombination, and DNA repair. Thus, identification of a mitochondrial form of this enzyme provides a tool with which to dissect mammalian mitochondrial genome dynamics.
Mol Cell Biol 1999 May
PMID:The human DNA ligase III gene encodes nuclear and mitochondrial proteins. 1020 10

UV damage endonuclease (Uve1p) from Schizosaccharomyces pombe was initially described as a DNA repair enzyme specific for the repair of UV light-induced photoproducts and proposed as the initial step in an alternative excision repair pathway. Here we present biochemical and genetic evidence demonstrating that Uve1p is also a mismatch repair endonuclease which recognizes and cleaves DNA 5' to the mispaired base in a strand-specific manner. The biochemical properties of the Uve1p-mediated mismatch endonuclease activity are similar to those of the Uve1p-mediated UV photoproduct endonuclease. Mutants lacking Uve1p display a spontaneous mutator phenotype, further confirming the notion that Uve1p plays a role in mismatch repair. These results suggest that Uve1p has a surprisingly broad substrate specificity and may function as a general type of DNA repair protein with the capacity to initiate mismatch repair in certain organisms.
Mol Cell Biol 1999 Jul
PMID:A Uve1p-mediated mismatch repair pathway in Schizosaccharomyces pombe. 1037 19

Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. In higher eukaryotes, Rad51 protein is essential for cell viability. The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region. Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy. The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E. coli. NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies. However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface. Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51. Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain.
J Mol Biol 1999 Jul 09
PMID:The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA binding surface as revealed by NMR. 1039 Mar 47

When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site. To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.
J Mol Biol 1999 Jul 16
PMID:In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites. 1039 22

Protease negative mutant of Xanthomonas campestris pathovar glycine 8ra (prt-mutant) was constructed by mutagenesis employing artificial transposon Omegon-Km. Transposon delivery was conducted through diparental conjugation using X. campestris pathovar glycine 8ra as recipient and Escherichia coli S17-1 carrying pJFF 3500 plasmid as the donor. The frequency of transconjugation was found 1.9 x 10(-7) per recipient. Enzyme analysis indicated the presence of mutant with lower protease activity than that of the wild-type. Genetic analysis employing pulsed-field gel electrophoresis (PFGE) of the genomic DNA digested with AseI or SpeI restriction endonuclease could significantly differentiate X. campestris pathovar glycine 8ra prt from the wild-type parent. The 9.85 kb pLR omega 6 plasmid was constructed from the genomic DNA of the prt mutant after being digested with KpnI restriction endonuclease and ligated with T4 DNA ligase.
Mol Biotechnol 1999 Apr
PMID:Characterization of transposon-generated protease mutant of Xanthomonas campestris pathovar glycine 8ra. Enzyme activity, cloning, and mapping of flanking DNA. 1046 67

Interaction of DNA repair proteins with damaged DNA in eukaryotic cells is influenced by the packaging of DNA into chromatin. The basic repeating unit of chromatin, the nucleosome, plays an important role in regulating accessibility of repair proteins to sites of damage in DNA. There are a number of different pathways fundamental to the DNA repair process. Elucidation of the proteins involved in these pathways and the mechanisms they utilize for interacting with damaged nucleosomal and nonnucleosomal DNA has been aided by studies of genetic diseases where there are defects in the DNA repair process. Two of these diseases are xeroderma pigmentosum (XP) and Fanconi anemia (FA). Cells from patients with these disorders are similar in that they have defects in the initial steps of the repair process. However, there are a number of important differences in the nature of these defects. One of these is in the ability of repair proteins from XP and FA cells to interact with damaged nucleosomal DNA. In XP complementation group A (XPA) cells, for example, endonucleases present in a chromatin-associated protein complex involved in the initial steps in the repair process are defective in their ability to incise damaged nucleosomal DNA, but, like the normal complexes, can incise damaged naked DNA. In contrast, in FA complementation group A (FA-A) cells, these complexes are equally deficient in their ability to incise damaged naked and similarly damaged nucleosomal DNA. This ability to interact with damaged nucleosomal DNA correlates with the mechanism of action these endonucleases use for locating sites of damage. Whereas the FA-A and normal endonucleases act by a processive mechanism of action, the XPA endonucleases locate sites of damage distributively. Thus the mechanism of action utilized by a DNA repair enzyme may be of critical importance in its ability to interact with damaged nucleosomal DNA.
Prog Nucleic Acid Res Mol Biol 1999
PMID:DNA repair and chromatin structure in genetic diseases. 1050 34

Two mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at EcoRI sites in vivo. Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli. In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at non-cognate EcoRV sites in the bacterial genome. EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.
Mol Microbiol 1999 Sep
PMID:DNA nicks inflicted by restriction endonucleases are repaired by a RecA- and RecB-dependent pathway in Escherichia coli. 1051 Feb 29

Oxidative damage to DNA has been documented in cells isolated from subjects with diabetes. Herein, we evaluate the mechanism(s) that regulate the expression of the DNA repair enzyme XPD. CHO cells transfected with the human insulin receptor (CHO/HIRc) showed a threefold increase in the level of XPD mRNA when compared to control CHO/neo cells (P < 0.01). The addition of insulin to serum-starved cells led to an increase in XPD mRNA levels in both CHO/neo and CHO/HIRc cells, in a time and dose dependent fashion. Insulin acted primarily by inducing XPD transcription. Moreover, inhibition of protein synthesis by cyclohexamide induced a marked degradation of XPD mRNA levels in insulin treated cells. Site-directed mutagenesis of the tyrosine-kinase domain of the insulin receptor abolished the increase in XPD mRNA resulting from the transfection with wild type insulin receptors (P < 0.001). Western blot analysis of cell extracts from CHO/neo and CHO/HIRc cells revealed an increase in XPD counterpart protein was also induced by transfecting cells with the human insulin receptor. Evaluation of DNA damage by means of internucleosomal fragmentation showed a dramatic decrease in DNA fragmentation in CHO cells transfected with wild-type insulin receptor compared to control CHO/neo cells. DNA fragmentation was further decreased by the addition of insulin in the culture medium. In summary, our data indicates that activation of the insulin receptor plays an important role in the cellular response leading to repair of damaged DNA.
Mol Cell Endocrinol 1999 Nov 25
PMID:Signalling via receptor tyrosine kinase modulates the expression of the DNA repair enzyme XPD in cultured cells. 1061 8


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