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PubMed1999

Is involvement of the surgical margins of large loop excision of the cervical transformation zone specimens for cervical intraepithelial neoplasia III a risk factor for persistent cervical intraepithelial neoplasia?

We aimed to test the hypothesis that patients with cervical intraepithelial neoplasia (CIN) III treated by large loop excision of the cervical transformation zone (LLETZ) are more likely to suffer recurrence of cervical abnormalities if the CIN reaches the margins of the index specimen. Three hundred and forty-five women treated by LLETZ in 1991, and 1992, and in whom the histological diagnosis was CIN III, were studied. The reports were reviewed and the cases were categorised as completely excised, incompletely excised, or undetermined. Cytological follow-up, or in some cases histological follow-up in the same year and in each of the 5 following years was tabulated. Follow-up data were recorded from the same and also surrounding institutions. The chi-squared test was used to compare the differences in recurrence rates between the three groups at the various points in time. A small advantage was demonstrated for those cases where excision was complete. This was statistically significant in the third following year (P=0.022). The resection margin status of LLETZ specimens for CIN III has only a minor effect on the likelihood of recurrence. Nevertheless, recording the status may be important in case of unexpected invasion. The relative reassurance the information will provide when the lesion has been excised may also be seen as worthwhile.

Journal Article↗

PubMed1985

Inhibition of excision repair of DNA in u.v.-irradiated Escherichia coli by phenethyl alcohol.

Membrane-specific drugs such as procaine and chlorpromazine have been shown to inhibit excision repair of DNA in u.v.-irradiated E. coli. One possible mechanism is that, if association of DNA with the cell membrane is essential for excision repair, this process may be susceptible to drugs affecting the structure of cell membranes. We examined the effect of phenethyl alcohol, which is a membrane-specific drug and known to dissociate the DNA-membrane complex, on excision repair of DNA in u.v.-irradiated E. coli cells. The cells were irradiated with u.v. light and then held at 30 degrees C in buffer (liquid-holding) in the presence or absence of phenethyl alcohol. It was found that phenethyl alcohol inhibits the liquid-holding recovery in both wild-type and recA strains, corresponding to its dissociating action on the DNA-membrane complex. Thus, the association of DNA with cell membrane is an important factor for excision repair in E. coli. Procaine did not show the dissociating effect, suggesting that at least two different mechanisms are responsible for the involvement of cell membrane in excision repair of DNA in E. coli.

Cell Membrane↗

PubMed1992

Introns excised from the Delta primary transcript are localized near sites of Delta transcription.

Introns excised from the primary transcript of Delta (Dl), a Drosophila neurogenic gene, accumulate to unusually high levels in embryos. High resolution in situ hybridization reveals a striking localization of the excised introns to two foci per embryonic nucleus. The number of foci can be altered by varying the number of Dl genes present in the embryonic nucleus, suggesting that the excised introns are localized near sites of Dl transcription. This conclusion is supported by the observation that larval and imaginal disc nuclei containing two copies of Dl exhibit only one focus of intron accumulation, as expected for nuclei in which homologous chromosomes are paired. Interestingly, the excised introns do not appear to diffuse away from the foci until late prophase, at which time the foci disperse into numerous small dots of hybridization. These results suggest that the excised Dl introns may be associated with a structural element within the nucleus that is dissociated during cell division.

Animals↗

PubMed1987

A comparison of calcium-activated potassium channel currents in cell-attached and excised patches.

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.

Calcium↗

PubMed1986

Analysis of DNA repair in XP-HeLa hybrids; lack of correlation between excision repair of u.v. damage and adenovirus reactivation in an XP(D)-like cell line.

Hybrids formed between HeLa cells and fibroblasts from xeroderma pigmentosum group D show either HeLa sensitivity or XPD-like hypersensitivity to u.v. radiation and corresponding high or low excision repair capability. Hybrids with low repair are presumed to have lost, via chromosome segregation, the HeLa wild type D alleles. In this paper we analyse the u.v. sensitivity and excision repair capability of another hybrid, HD1A, derived spontaneously from the normally sensitive hybrid HD1. While HD1A closely resembles the XPD phenotype in terms of u.v. sensitivity and excision repair it differs from XPD because of its ability to reactivate u.v.-irradiated adenovirus 2 to an extent similar to that of its HeLa parent. This capacity functionally dissociates excision repair of chromatin-based damage from damage in a viral environment. Moreover, on the basis of complementation studies the excision repair of genomic damage by HD1A is subtly different from that of a true XPD-like hybrid, HD2. The data are discussed in terms of a second change in the defective D allele of the HD1A cell.

Adenoviridae↗

PubMed1986

Proliferation-dependent regulation of base excision repair in normal and SV-40 transformed human cells.

The effect of SV-40 viral transformation of human cells on the proliferation-dependent regulation of base excision repair was examined. Normal human diploid WI-38 fibroblasts and SV-40 transformed WI-38 cells were used to examine whether the process of transformation perturbed the regulation of base excision repair. The regulation of excision repair enzymes in each cell type was examined by quantitating: the specific activities of the base excision repair enzymes uracil DNA glycosylase and hypoxanthine DNA glycosylase as a function of cell growth; and immunological analysis of the uracil DNA glycosylase using monoclonal antibodies to human uracil DNA glycosylase. In each cell type, both DNA glycosylase activities were increased as a function of cell proliferation; the extent of enhancement being greater in the transformed cells. As defined by ELISA and immunoblot analysis, the induced uracil DNA glycosylases in both cell types share the same determinants. Although other repair processes are altered upon SV-40 transformation, these results suggest that the proliferation-dependent regulation of these base excision repair enzymes is retained in SV-40 transformed human cells.

Cell Division↗

PubMed1987

Action of etoposide (VP-16-123) on human cells: no evidence for topoisomerase II involvement in excision repair of u.v.-induced DNA damage, nor for mitochondrial hypersensitivity in ataxia telangiectasia.

The excision repair of u.v. damage has been supposed to involve an initial action of DNA topoisomerase II, since some pre-incision step is sensitive to novobiocin, a topoisomerase II inhibitor. But novobiocin also affects mitochondrial structure and ATP metabolism, and this may account for its apparent inhibition of energy-dependent excision repair. We have investigated the effects of etoposide, another inhibitor of topoisomerase II, on u.v.-irradiated human cells: it is a more specific agent with no immediate side-effects on mitochondria. But etoposide is without effect on cellular excision repair, at the pre-incision stage or at the later stages of either DNA resynthesis or strand break ligation; nor does it potentiate cell killing after u.v. irradiation. The chromosome decondensation that accompanies incomplete excision repair in mitotic cells is likewise not greatly affected by etoposide. Therefore, if topoisomerase II is involved in excision repair or its regulation, it acts through a process that in whole cells is insensitive to etoposide. In ataxia telangiectasia cells, which are known to be hypersensitive to etoposide, the mitochondrial activities are not abnormally affected.

Ataxia Telangiectasia↗

PubMed2002

DNA base excision repair of uracil residues in reconstituted nucleosome core particles.

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.

Animals↗

PubMed1996

The hobo transposable element excises and has related elements in tephritid species.

Function of the Drosophila melanogaster hobo transposon in tephritid species was tested in transient embryonic excision assays. Wild-type and mutant strains of Anastrepha suspensa, Bactrocera dorsalis, B. cucurbitae, Ceratitis capitata, and Toxotrypana curvicauda all supported hobo excision or deletion both in the presence and absence of co-injected hobo transposase, indicating a permissive state for hobo mobility and the existence of endogenous systems capable of mobilizing hobo. In several strains hobo helper reduced excision. Excision depended on hobo sequences in the indicator plasmid, though almost all excisions were imprecise and the mobilizing systems appear mechanistically different from hobo. hobo-related sequences were identified in all species except T. curvicauda. Parsimony analysis yielded a subgroup including the B. cucurbitae and C. capitata sequences along with hobo and Hermes, and a separate, more divergent subgroup including the A. suspensa and B. dorsalis sequences. All of the sequences exist as multiple genomic elements, and a deleted form of the B. cucurbitae element exists in B. dorsalis. The hobo-related sequences are probably members of the hAT transposon family with some evolving from distant ancestor elements, while others may have originated from more recent horizontal transfers.

Amino Acid Sequence↗

PubMed1998

A mutation in Paramecium tetraurelia reveals functional and structural features of developmentally excised DNA elements.

The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28-882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tcl transposons (KLOBUTCHER and HERRICK 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES259 I has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its Mild-type micronuclear copy through multiple sexual generations.

Animals↗

PubMed1986

The different intron 2 species excised in vivo from the E2A premRNA of adenovirus-2: an approach to analyse alternative splicing.

In the early period of cellular infection by adenovirus 2, the E2A region gives rise to 2 major mRNA species of 2.0 and 2.3 kilobases, formed by alternative excisions of intron 2 (Gattoni et al., 1986, J. Mol. Biol. 187, 379-307). We have analysed the excision pathways of this intron. Two major intron species of 626 and 337 nucleotides, generated by the use of 2 consensus 3' splicing sites and a minor intron species of 520 nucleotides, generated by the use of another weaker 3' splicing site, are identified, the 3 species sharing a common 5' splicing site. They are detected predominantly in the lariat form. For the 2 major species we analyzed, the branched nucleotides are localized at consensus branching sequences, 26 or 25 nucleotides upstream from the 3' terminal AG. Our results confirm that the first reactions of cleavage at the 5' end of introns and branching occur in vivo as described in in vitro systems. The second predominant form of intron 2 is the linear segment, whereas the nicked lariat form which is very minor, might not be a genuine product of in vivo splicing. All intron 2 molecules show practically intact 5' and 3' terminal sequences, indicating that they are well protected against nuclease attack throughout their life. Therefore, these results indicate that the primary reaction following the excision of the lariat intron is debranching. In addition, the existence of a potential 5' splicing site contiguous to the major internal 3' splicing site raised the possibility of an elimination of the major 626 nucleotide intron in 2 cycles of excision. However, we demonstrate that intron 2 is systematically excised by a one cycle process, which is likely to represent the general rule for the production of correctly spliced mRNA.

Adenoviruses, Human↗

PubMed1991

High fidelity developmental excision of Tec1 transposons and internal eliminated sequences in Euplotes crassus.

Following the sexual phase of its life cycle, the hypotrichous ciliate Euplotes crassus transforms a copy of its chromosomal micronucleus into a transcriptionally active macronucleus containing short, linear, gene-sized DNA molecules. Tens of thousands of DNA breakage and joining, or splicing, events occur during macronuclear development. The DNA removed by such events includes transposon-like elements, referred to as Tec1 elements, as well as segments of unique sequence DNA, termed internal eliminated sequences (IESs). Both types of elements are bounded by short direct repeats. In the current study, a polymerase chain reaction (PCR) and DNA sequencing strategy has been used to examine the fidelity of excision of two Tec1 elements and three IESs. In all cases, the vast majority of excision events were found to be precise, with one copy of the terminal direct repeats retained at the empty site in the macronuclear DNA molecule. These results, in combination with previous studies that have characterized the excised DNA elements, indicate that the two products of excision (the free element and the macronuclear DNA molecule) share DNA sequences. This suggests that excision events are initiated by staggered cuts in the chromosomal DNA.

Animals↗

PubMed1992

Purification of PCNA as a nucleotide excision repair protein.

Human cell free extracts carry out nucleotide excision repair in vitro. The extract is readily separated into two fractions by chromatography on a DEAE column. Neither the low salt (0.1 M KCl) nor the high salt (0.8 M KCl) fractions are capable of repair synthesis but the combination of the two restore the repair synthesis activity. Using the repair synthesis assay we purified a protein of 37 kDa from the high salt fraction which upon addition to the low salt fraction restores repair synthesis activity. Amino acid sequence analysis, amino acid composition and immunoblotting with PCNA antibodies revealed that the 37 kDa protein is the proliferating cell nuclear antigen (PCNA) known to stimulate DNA Polymerases delta and epsilon. By using an assay which specifically measures the excision of thymine dimers we found that PCNA is not required for the actual excision reaction per se but increases the extent of excision by enabling the excision repair enzyme to turn over catalytically.

Amino Acid Sequence↗

PubMed1993

Reconstitution of mammalian excision repair activity with mutant cell-free extracts and XPAC and ERCC1 proteins expressed in Escherichia coli.

Nucleotide excision repair in humans involves the coordinated actions of 8-10 proteins. To understand the roles of each of these proteins in excision it is necessary to develop an in vitro excision repair system reconstituted entirely from purified proteins. Towards this goal we have expressed in E. coli two of the 8 genes known to be essential for the excision reaction. XPAC and ERCC1 were expressed as fusion proteins with the Escherichia coli maltose binding protein (MBP) and purified to > 80% homogeneity by affinity chromatography. The purified proteins either as fusions or after cleavage from the MBP were able to complement the CFE of cells with mutations in the corresponding genes in an excision assay with thymine dimer containing substrate.

ATP-Binding Cassette Transporters↗

PubMed1994

Precise excision of the retrotransposon gypsy from the forked and cut loci in a genetically unstable D. melanogaster strain.

The genetically unstable Mutator Strain of D. melanogaster is characterised by a high frequency of spontaneous mutations and their reversions. Three forked mutants were obtained independently and several reversions arose spontaneously with frequency of 10(-3)-10(-4). The sites of integration and excision of the gypsy retrotransposon were analysed by Southern blot analysis and sequencing of PCR fragments. In all cases gypsy had inserted at the end of the third exon of the major transcript of the forked gene, causing the duplication of TCCA target sequence. All the reversions resulted from precise excision of the gypsy. A double mutant containing ct6 and f1, caused by gypsy insertions into untranslated regions of the corresponding genes, was constructed. Two spontaneous ct6f+ revertants as well as one ct+f1 revertant were obtained from this line. Sequence analysis of gypsy integration and excision sites revealed that in all cases gypsy excision was also precise. These experiments constitute the first demonstration of precise excision of LTR-containing elements from their host genomes.

Animals↗

PubMed1996

Another heritage from the RNA world: self-excision of intron sequence from nuclear pre-tRNAs.

The intervening sequences of nuclear tRNA precursors are known to be excised by tRNA splicing endonuclease. We show here that a T7 transcript corresponding to a pre-tRNA(Tyr) from Arabidopsis thaliana has a highly specific activity for autolytic intron excision. Self-cleavage occurs precisely at the authentic 3'-splice site and at the phosphodiester bond one nucleotide downstream of the authentic 5'-splice site. The reaction results in fragments with 2',3'-cyclic phosphate and 5'-OH termini. It is resistant to proteinase K and/or SDS treatment and is not inhibited by added tRNA. The self-cleavage depends on Mg2+ and is stimulated by spermine and Triton X-100. A set of sequence variants at the cleavage sites has been analysed for autolytic intron excision and, in parallel, for enzymatic in vitro splicing in wheat germ S23 extract. Single-stranded loops are a prerequisite for both reactions. Self-cleavage not only occurs at pyrimidine-A but also at U-U bonds. Since intron self-excision is only about five times slower than the enzymatic intron excision in a wheat germ S23 extract, we propose that the splicing endonuclease may function by improving the preciseness and efficiency of an inherent pre-tRNA self-cleavage activity.

Arabidopsis↗

PubMed1996

Characterization of the DNA polymerase requirement of human base excision repair.

Base excision repair is one of the major mechanisms by which cells correct damaged DNA. We have developed an in vitro assay for base excision repair which is dependent on a uracil-containing DNA template. In this report, we demonstrate the fractionation of a human cell extract into two required components. One fraction was extensively purified and by several criteria shown to be identical to DNA polymerase beta (Polbeta). Purified, recombinant Polbeta efficiently substituted for this fraction. Escherichia coli PolI, mammalian Poldelta and to a lesser extent Polalpha and epsilon also functioned in this assay. We provide evidence that multiple polymerases function in base excision repair in human cell extracts. A neutralizing antibody to Polbeta, which inhibited repair synthesis catalyzed by pure Polbeta by approximately 90%, only suppressed repair in crude extracts by a maximum of approximately 70%. An inhibitor of Polbeta, ddCTP, decreased base excision repair in crude extracts by approximately 50%, whereas the Polalpha/delta/epsilon inhibitor, aphidicolin, reduced the reaction by approximately 20%. A combination of these chemical inhibitors almost completely abolished repair synthesis. These data suggest that Polbeta is the major base excision repair polymerase in human cells, but that other polymerases also contribute to a significant extent.

Chromatography, DEAE-Cellulose↗

PubMed1997

Excision of a conjugative transposon in vitro by the Int and Xis proteins of Tn916.

The roles of purified Int and Xis proteins of the conjugative transposon Tn 916 in excision of a deletion derivative of the closely related element Tn 1545 were investigated. At a low salt concentration (37.5 mM NaCl), Int alone was able to promote limited excision to produce a covalently closed circular form of the transposon, showing that Tn 916 Int can catalyze both DNA cleavage and strand exchange. This reaction was stimulated by Xis. At higher salt concentrations (150 mM NaCl), excision by Int alone was reduced to barely detectable levels and Xis was required for excision. The low salt, Xis-stimulated reaction was approximately 8-fold more efficient than the high salt, Xis-dependent reaction. These results reflect in vivo requirements for Int and Xis in excision.

Amino Acid Sequence↗