Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.25.1 (deoxyribonuclease)
1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. (i) In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. (ii) In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Cairns-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. (iii) Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules. These results are in agreement with and reinforce the model in which UV lesions are a barrier to the replication fork movement when present in the template for the leading strand; when lesions are in the template for the lagging strand they inhibit synthesis or completion of Okazaki fragments, leaving gaps opposite the lesion. Moreover, cellular DNA repair-linked endonucleolytic activity may induce double-stranded breaks in the blocked region of the replication forks, resulting in the tailed structures observed in viral DNA molecules obtained from excision repair-proficient cell lines.
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PMID:Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands. 284 36

A structural gene for T4 endonuclease V was constructed by ligating synthetic oligonucleotides. The endonuclease V was overproduced in E. coli under control of the E. coli tryptophan promoter and purified to apparent homogeneity. The product had comparable DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease activities to the natural enzyme in vitro. When this endonuclease V was microinjected into the cytoplasm of xeroderma pigmentosum (XP) cells of complementation group A, B, C, D, F, G or H, unscheduled DNA synthesis (UDS) above the residual level was detected in all the cells at a dose of about 10(3) molecules following UV irradiation. The gain numbers of UDS in these XP cells increased with increase in the dose of enzyme and reached a plateau at the normal cell level on introduction of about 10(4) molecules. Introduction of more enzyme into either XP cells or normal human cells did not increase the grain number under regular labelling conditions (2.5 h, 37 degrees C). In normal mouse cells, introduction of the enzyme increased the grain number more than 4-fold under the same conditions during at least 8.5 h following UV irradiation. Furthermore, with a labelling time of 30 min, the enzyme more than doubled the grain number even in normal human cells.
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PMID:Microinjection of T4 endonuclease V produced by a synthetic denV gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells. 291 66

We have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. We studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines (D0 = 56 J X m-2) than in the other human cell lines tested (normal, ataxia-telangiectasia, Lesch-Nyhan, retinoblastoma)(D0 = 680 J X m-2)(D0 is the dose that reduces the percentage of CAT activity by 63% along the exponential portion of the dose-response curve). The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA.
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PMID:One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. 299 75

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.
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PMID:DNA repair synthesis in human fibroblasts requires DNA polymerase delta. 333 6

The damage-directed strand incision step in the nucleotidyl DNA excision-repair pathway (NDERP) was characterized in quiescent monolayer cultures of human fibroblasts in which the plasma membrane was selectively permeabilized with saponin. When permeable normal human fibroblasts (NHF) were incubated in a DNA-repair assay mixture lacking the deoxyribonucleoside triphosphate precursors, the numbers of UV-dependent DNA-strand breaks were increased by about 9-fold consistent with the uncoupling of incision from gap-filling DNA synthesis and ligation. In uncoupled NHF omission of ATP reduced the numbers of UV-dependent strand breaks by 84% confirming the requirement for ATP for reparative strand incision. Time-course experiments indicated that the maximum rate of strand incision occurred in the first 10 min of incubation of permeable cells and diminished to 16-28% of this rate between 30 and 60 min of incubation. The initial rate of incision in permeable NHF was estimated to be 20% of that seen in intact fibroblasts. Dose-response studies indicated an initial saturation of strand incision activity at fluences between 10 and 25 J/m2. In permeable group A xeroderma pigmentosum fibroblasts (XPA) few UV-dependent incisions were produced after 10-25 J/m2. In the xeroderma pigmentosum variant (XPV) strain that we studied, strand incisions saturated at a plateau level that was about twice that seen in the NHF strain suggesting the preservation of a higher level of incision activity after permeabilization. After fluences above 50 J/m2 additional strand incision was observed in all cell strains reflecting the activity of a damage-dependent endodeoxyribonuclease that is independent of the NDERP. Saponin-treated fibroblasts were also permeable to pancreatic deoxyribonuclease I and the UV-DNA endonuclease from M. luteus indicating that these preparations may be used for in vitro complementation.
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PMID:Reparative strand incision in saponin-permeabilized human fibroblasts. 367 Mar 27

The UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of the UV-induced UDS, in some cells to the repair-proficient human level, was observed. Another prokaryotic DNA-repair enzyme, T4 endonuclease V, restored the UV-induced UDS in a similar way after microinjection into XP cells. Since both enzymes specifically catalyse only the incision of UV-irradiated DNA, we conclude that this activity is impaired in cells of all 9 excision-deficient XP complementation groups tested.
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PMID:Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups. 383 45

DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair.
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PMID:Repair of ultraviolet radiation damage in xeroderma pigmentosum cells belonging to complementation group F. 720 91

A non-sensitive, 8-yr-old male patient (termed UV81KO) with only acute recurrent sunburns and without any other physical or neuromental retardations was studied. The patient's skin exhibited lowered minimal erythema doses between 280 and 300 nm monochromatic wavelengths without delayed peaking of erythema. UV81KO skin fibroblasts in culture was 5-fold more sensitive to 254 nm UV killing than normal cells, though the response of obligatory heterozygotes was normal. UV81KO cells were also more sensitive to killings by fluorescent sunlamp (295-300 nm UV-B) radiation, 4-nitroquinoline-1-oxide, and N-hydroxy-acetyl aminofluorene, but not by monofunctional decarbamoyl mitomycin C, bifunctional mitomycin C, and alkylating agents (methyl methanesulfonate, ethyl methanesulfonate, N-methyl-N-nitrosourea). Assays for unscheduled DNA synthesis, T4 endonuclease V-susceptible sites (pyrimidine dimers), endogenous excision-break accumulation by arabinofuranosyl cytosine-plus-hydroxyurea, single-strand-break rejoining, and molecular-weight increase of pulse-chased DNA in irradiated cells indicated no apparently detectable defects in nucleotide-excision repair processes and in replicative bypass in UV81KO cells. Despite the repair proficiency as such, UV81KO cells showed the defective recovery of DNA synthesis after 254 nm UV irradiation with 1 and 5 J/m2, at which dose the recovery occurred in normal cells. The base line level of sister-chromatid exchanges (SCEs) was higher in UV81KO cells (10-12 SCEs/cell) than in normal cells (5 SCEs/cell), although the induction rate of SCEs by 254 nm UV in UV81KO cells was the same as in normal cells. Such clinical, cellular and molecular characteristics and comparison to those in the other photodermatoses (xeroderma pigmentosum, Cockayne's syndrome, the 11961 disorder, Bloom's syndrome) can make a clear distinction of UV81KO from the others. Thus, the UV81KO disorder is put forward as a new photodermatosis with a defect in the recovery of post-UV DNA synthesis.
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PMID:A new human photosensitive subject with a defect in the recovery of DNA synthesis after ultraviolet-light irradiation. 726 57

The repair of cyclobutane pyrimidine dimers was measured under non-replicating conditions in a 2054-bp fragment of a cAMP-inducible cysteine proteinase (CP2) gene in D. discoideum. Overall genomic repair was unaffected by cAMP. The removal of dimers from CP2 in the wild-type NP2 cells as quantified using T4 endonuclease V was independent of transcription and was the same as in the overall genome. In a UV-sensitive radC mutant in which the rate of overall dimer removal was previously shown to be reduced, the initial rate of dimer removal in the uninduced CP2 gene (-cAMP) was 3-fold lower compared to the induced gene (+cAMP), in which repair was identical to that for the induced and uninduced states of NP2. D. discoideum may have two pathways for repairing dimers. One, effective in the wild-type strain but of reduced efficiency in radC repairs dimers equally well independent of transcription and at about the same rate as in the overall genome. A second pathway, retained in radC, repairs dimers more slowly in the overall genome and in the uninduced CP2 gene while undergoing the wild-type rate of repair in the transcriptionally active gene. Hence radC has reduced ability to repair transcriptionally inactive DNA, a defect similar to that of xeroderma pigmentosum group C.
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PMID:Differential repair of UV damage in a developmentally regulated gene of Dictyostelium discoideum. 751 Mar 68

A shuttle vector (pZH-1) carrying the E. coli lacZ gene under control of the SV40 early promoter was irradiated with UV and introduced into repair-proficient or repair-deficient human cell lines. The expression of irradiated lacZ compared to unirradiated lacZ was greater in repair-proficient cells (HT-1080) than in repair-deficient cells (XP12RO-SV40) belonging to xeroderma pigmentosum complementation group A. To ascertain whether the expression of lacZ in the repair-proficient cells was correlated with the removal of cyclobutane pyrimidine dimers (CPDs), we purified DNA from the recipient cells and used the CPD-specific enzyme T4 endonuclease V to measure the frequency of CPDs remaining in the plasmid as a whole and in two restriction fragments derived from it. We found that removal of CPDs occurred in both fragments in the repair-proficient cells but not in the repair-deficient cells. Our results provide the first direct evidence for the removal of CPDs from UV irradiated plasmids introduced into human cells and support the notion that expression of the UV-damaged lacZ gene in repair-proficient human cells reflects the removal of transcription blocking lesions from the gene.
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PMID:Removal of cyclobutane pyrimidine dimers from a UV-irradiated shuttle vector introduced into human cells. 794 23


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