Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DNA extracted from xeroderma pigmentosum human fibroblasts previously irradiated with 12.5 J/m2 of UV light and pulse-labeled for 45 min with radioactive and (or) heavy precursors, was used to determine the structural characteristics of the replication fork. Density equilibrium centrifugation experiments showed that a fork moved 6 micrometer in 45 min and bypassed 3 pyrimidine dimers in both strands. The same length was covered in 15-20 min in control cells. The delay in irradiated cells was apparently due to pyrimidine dimers acting as temporary blocks to the fork movement. Evidence for this interpretation comes from kinetics of incorporation of [3H]thymidine into DNA, which show that the time necessary to attain a new stable level of DNA synthesis in irradiated cells is equivalent to that required for the replication fork to cover the interdimer distance in one strand. On the other hand, the action of S1 nuclease on DNA synthesized soon after irradiation gives rise to a bimodal distribution in neutral sucrose gradients, one peak corresponding to 43 X 10(6) daltons and the other to 3 X 10(6) daltons. These two DNA species are generated by the attack of the S1 nuclease on single-stranded regions associated with the replication fork. A possible explanation for these results is given by a model according to which there is a delayed bypass of the dimer in the leading strand and the appearance of gaps opposite pyrimidine dimers in the lagging strand, as a direct consequence of the discontinuous mode of DNA replication. In terms of the model, the DNA of 43 X 10(6) daltons corresponds to the leading strand, linked to the unreplicated branch of the forks, whereas the piece of 3 X 10(6) daltons is the intergap DNA coming from the lagging strand. Pulse and chase experiments reveal that the low molecular weight DNA grows in a pattern that suggests that more than one gap may be formed per replication fork.
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PMID:Structure of the replication fork in ultraviolet light-irradiated human cells. 23 82

Monoadducts and cross-links formed in DNA of human cells by a psoralen derivative, 4'-hydroxy-methyl-4,5',8-trimethylpsoralen (HMT), have been measured by a new, simple method, based on S1 nuclease digestion of 3H-labeled adducts in DNA, that provides rapid information on the repair of both classes of lesions. Normal human fibroblasts and cells from patients with dyskeratosis congenita and xeroderma pigmentosum (XP) group C were capable of removing both monoadducts and cross-links, whereas XP groups A and D failed to remove either. An XP revertant, isolated from a group A cell line on the basis of an acquired mutagen-induced resistance to ultraviolet light, has the unique property of being capable of removing cross-links but not monoadducts. Consistent with this property, the XP revertant was found to be resistant to cell killing by the cross-linking psoralen derivative, HMT, but as sensitive as its parental cell line to a monofunctional psoralen derivative, 5-methylisopsoralen.
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PMID:Unique cross-link and monoadduct repair characteristics of a xeroderma pigmentosum revertant cell line. 282 32

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

Native newly synthesized DNA from human cells (xeroderma pigmentosum type) irradiated with ultraviolet light releases short pieces of DNA (L-DNA) when incubated with the single-strand specific S1 nuclease. This is not observed in the case of unirradiated cells. Previous experiments had shown that the L-DNA resulted from the action of S1 nuclease upon gaps, i.e., single-stranded DNA discontinuities in larger pieces of double-stranded DNA. We verified that the duplex L-DNA, that arises from the inter-gap regions upon S1 nuclease treatment, has a size which approximates the distance between two pyrimidine dimers on the same strand; this has been observed at different fluences of ultraviolet-light and indicates that the gap is related to or opposite the dimer. A method was devised to measure the size of the gaps. A Poisson distribution analysis of the percentage of the L-DNA produced as a function of S1 nuclease concentration made this possible. 65% of the gaps corresponded to stretches of 1,250 nucleotides and 35% to stretches of 150 nucleotides. These parameters have been considered in the proposition of a model for DNA synthesis on a template containing pyrimidine dimers.
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PMID:Size and frequency of gaps in newly synthesized DNA of xeroderma pigmentosum human cells irradiated with ultraviolet light. 626 12

Inhibition of the gap-filling, polymerizing step of excision repair by 1-beta-D-arabinofuranosylcytosine (ara-C) after irradiation with ultraviolet light in human diploid fibroblasts resulted in the formation of persistent DNA strand breaks in G1, G2, and plateau phase cells, but not in S phase cells. Addition of hydroxyurea to ara-C resulted in partial inhibition of repair in S phase cells. These observations can be explained either in terms of changing roles in repair for different DNA polymerases throughout the cell cycle or by the presence of a pool of deoxycytidine nucleotides during S phase equivalent to be an external source of deoxycytidine at 50 microM concentration. A similar concentration dependence on ara-C was observed for inhibition of repair in normal human, xeroderma pigmentosum (XP) variant, and Cockayne's syndrome cells but slightly more in XP variant cells. Exonuclease III and S1 nuclease independently both degraded about 50% of the 3H-thymidine incorporated into repaired regions in the presence of ara-C. Sequential digestion with both enzymes degraded nearly 90% of the repaired regions. These observations can be explained if excision repair proceeds by displacing the damaged strand so that both the 3H-labeled patch and the damaged region are still ligated to high molecular weight DNA and compete for the same complementary strand during in vitro incubation with the nucleases. The amount of 3H-thymidine incorporated in DNA by repair decreased with increasing concentrations of ara-C and hydroxyurea, suggesting that the incomplete patches became shorter under these conditions. Extrapolation of the digestion kinetics with exonuclease III permits an estimate of the normal patch size of about 100 nucleotides, consistent with previous estimates.
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PMID:Sensitivity of excision repair in normal human, xeroderma pigmentosum variant and Cockayne's syndrome fibroblasts to inhibition by cytosine arabinoside. 726 70

Photoactivated 8-methoxypsoralen (8-MOP) has been proven to be clinically effective for a number of dermatological conditions including lichen planus, mycosis fungoides, and psoriasis. 8-MOP forms two types of covalent photoproducts with DNA, monoadducts, and bifunctional adducts which cross-link the two DNA strands. Angelicin is a congener of 8-MOP which forms only monoadducts. We have used the combined density and isotopic labeling technique to study repair replication in cultured human fibroblasts treated with either of these compounds and exposed to near-ultraviolet light. In human diploid fibroblasts (WI-38), the time course of repair replication for both compounds is similar. Drug concentration and ultraviolet dose responses are also similar for 8-MOP and angelicin. No repair replication was stimulated by either compound in xeroderma pigmentosum cells from Complementation Group A (XP12BE). These results suggest that repair replication in response to 8-MOP is primarily a response to monoadducts and that the enzymatic pathway for this repair synthesis shares at least one step with the pathway for repair of pyrimidine dimers. Cross-link persistence in treated cells was assayed by use of the single-strand-specific S1 nuclease to digest DNA that did not renature readily following heat denaturation. Partial removal of cross-links was observed in normal, xeroderma pigmentosum variant, and Fanconi's anemia fibroblasts, but not in xeroderma pigmentosum Group A cells.
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PMID:DNA repair in human cells containing photoadducts of 8-methoxypsoralen or angelicin. 747 Oct 88