共查询到20条相似文献,搜索用时 31 毫秒
1.
S. A. Nedospasov A. N. Shakhov B. Hirt 《Journal of biomolecular structure & dynamics》2013,31(5):907-913
Abstract We have analysed SI sensitivity of SV40 minichromosomes isolated from the nuclei of infected cells at the late stage of infection. We show that a fraction of purified minichromosomes is sensitive towards double-strand cleavage by SI nuclease. The pattern of specific cleavage reminiscent ofthat found for subcloned fragment under supercoiling is superimposed upon apparently random double-strand cuts along the entire regulatory region. Therefore, the cleavage sites are not exclusively confined to the regions with the reported alternate DNA conformation. 相似文献
2.
'Sliding' of nucleosomes along DNA under nearly physiological conditions was studied using treatment of SV 40 minichromosomes with the single-cut restriction endonucleases EcoRI and BamHI. Each enzyme can convert no more than 20-25% of the circular DNA molecules of minichromosomes into the linear form irrespective of the presence of histone H1. This suggests absence of the nucleosomes lateral migration (sliding) along DNa at least in the vicinity of the restriction endonucleases cleavage sites during several hours of incubation. The sites available for EcoRI and BamHI in minichromosomes seem to be located predominantly in the spacer DNA regions of nucleosomes. Introduction of only one double-strand (but not single-strand) break into the DNA of minichromosomes stripped of histone H1 is sufficient to induce redistribution of the nucleosome core particles due to their sliding along DNA. Thus, sliding of the nucleosome core particles can be induced under physiological conditions by rather low energy expenditures. 相似文献
3.
Simian virus 40 minichromosomes contain torsionally strained DNA molecules. 总被引:7,自引:1,他引:6
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed. 相似文献
4.
Activation of poly(adenosine diphosphate ribose) polymerase by SV 40 minichromosomes: effects of deoxyribonucleic acid damage and histone H1 总被引:3,自引:0,他引:3
Poly(ADP-ribose) polymerase is a chromosomal enzyme that is completely dependent on added DNA for activity. The ability of DNA molecules to activate the polymerase appears to be enhanced by the presence of DNA damage. In the present study, we used SV 40 DNA and SV 40 minichromosomes to determine whether different types of DNA damage and different chromosomal components affect stimulation of polymerase activity. Treatment of SV 40 minichromosomes with agents or conditions that induced single-strand breaks increased their ability to stimulate poly(ADP-ribose) synthesis. This stimulation was enhanced by addition of histone H1 at a ratio of 1 microgram of histone H1 to 1 microgram of DNA. Higher ratios of histone H1 to DNA suppressed the ability of SV 40 minichromosomes containing single-strand breaks to stimulate enzyme activity. Treatment of SV 40 minichromosomes or SV 40 DNA with HaeIII restriction endonuclease to produce double-strand breaks markedly stimulated poly(ADP-ribose) polymerase activity. The stimulation of poly(ADP-ribose) polymerase by double-strand breaks occurred in the absence of histone H1 and was further enhanced by adding histone H1 up to ratios of 2 to 1 relative to DNA. At higher ratios of histone H1 to DNA, the presence of the histone continued to enhance the poly(ADP-ribose) synthesis stimulated by double-strand breaks. 相似文献
5.
Cleavage of ultraviolet light-irradiated DNA by single strand-specific S1 endonuclease 总被引:3,自引:0,他引:3
It was evidenced that the single strand-specific S1 endonuclease could cleave the ultraviolet light-irradiated T7 DNA. The cleavage of ultraviolet light-irradiated T7 DNA by S1 endonuclease was studied by sucrose density gradient centrifugation. The extent of cleavage was proportional to the dose of ultraviolet light given, the concentration of endonuclease and the ionic strength in the reaction. The cleavage consisted of both single-strand and double-strand breaks. The double-strand breaks were observed even at relatively lower dose of ultraviolet light. It seems likely that S1 endonuclease can recognize the alteration in the double-helical structure produced by ultraviolet light-irradiation rather than specifically attack ultraviolet light-induced photoproducts. 相似文献
6.
7.
Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S1 nuclease 总被引:4,自引:0,他引:4
Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S1 nuclease cleavage. The S1-cleavage occurred once at these sites. In the absence of added Mg2+, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S1, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S1-cleavable, unbasepaired sites. 相似文献
8.
9.
We have developed an assay in S. cerevisiae in which clones of cells that contain intact dicentric minichromosomes are visually distinct from those that have rearranged to monocentric minichromosomes. We find that the instability of dicentric minichromosomes is apparently due to mitotic nondisjunction accompanied by occasional structural rearrangements. Monocentric minichromosomes arising by rearrangement of the plasmid are rapidly selected in the population since dicentric minichromosomes depress the rate of cell division. We show that the ability of one centromere to compete with another in dicentric minichromosomes requires the presence of both of the conserved structural elements, CDE II and CDE III. Dicentric minichromosomes can be stabilized if one of the centromeres on the molecule is functionally hypomorphic because of mutations in CDE II even though these mutant centromeres are highly efficient in monocentric molecules. Stable dicentric molecules can also be produced by decreasing the space between two wild-type centromeres on the same molecule. These results suggest plausible pathways for changes in chromosome number that accompany evolution. 相似文献
10.
Phosphorylation of a yeast histone H2A at C-terminal serine 129 has a central role in double-strand break repair. Mimicking H2A phosphorylation by replacement of serine 129 with glutamic acid (hta1-S129E) suggested that phosphorylation destabilizes chromatin structures and thereby facilitates the access of repair proteins. Here we have tested chromatin structures in hta1-S129 mutants and in a C-terminal tail deletion strain. We show that hta1-S129E affects neither nucleosome positioning in minichromosomes and genomic loci nor supercoiling of minichromosomes. Moreover, hta1-S129E has no effect on chromatin stability measured by conventional nuclease digestion, nor does it affect DNA accessibility and repair of UV-induced DNA lesions by nucleotide excision repair and photolyase in vivo. Similarly, deletion of the C-terminal tail has no effect on nucleosome positioning and stability. These data argue against a general role for the C-terminal tail in chromatin organization and suggest that phosphorylated H2A, gamma-H2AX in higher eukaryotes, acts by recruitment of repair components rather than by destabilizing chromatin structures. 相似文献
11.
12.
Effect of X-ray induced DNA damage on DNAase I hypersensitivity of SV40 chromatin: relation to elastic torsional strain in DNA. 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The effect of X-irradiation on DNAase I hypersensitivity of SV40 minichromosomes within nuclei or free in solution was investigated. The susceptibility of the specific DNA sites in the control region of minichromosomes to DNAase I decreased in a dose dependent manner after irradiation of isolated nuclei. On the other hand, the irradiation of minichromosomes extracted from nuclei in 0.1 M NaCl-containing buffer almost did not affect the level of their hypersensitivity to DNAase I. This suggests that DNAase I hypersensitivity may be determined by two different mechanisms. One of them may be connected with elastic torsional strain within a fraction of minichromosomes and another seems to be determined by nucleosome free region. The first mechanism may be primarily responsible for the hypersensitivity of minichromosomes within nuclei. After irradiation of the intact cells, DNAase I hypersensitivity tested in nuclei substantially increased. This was connected with activation of endogeneous nucleases by X-irradiation which led to accumulation of single- and double-strand breaks superimposed to DNAase I induced breaks in the control region of SV40 DNA. 相似文献
13.
Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Strumberg D Pilon AA Smith M Hickey R Malkas L Pommier Y 《Molecular and cellular biology》2000,20(11):3977-3987
Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA. 相似文献
14.
15.
Cruciforms persist in reconstituted minichromosomes, as revealed by cleavage with specific nucleases and hybridization with synthetic oligonucleotides. Relaxation by topoisomerase I suggests that cruciforms are located mainly on internucleosomal DNA and that their persistence on minichromosomes may be due to kinetic effects. The analysis of the kinetic behaviour of cruciforms in minichromosomes shows a definite velocity of reabsorption with respect to stable cruciforms in supercoiled naked DNA. An explanation based on suppression of the untwisting of linker DNA due to adjacent nucleosomes is proposed. 相似文献
16.
Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron. 总被引:12,自引:1,他引:11
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Streptomyces lividans DNA contains a modification which makes it susceptible to double-strand cleavage during electrophoresis in buffers contaminated with ferrous iron (which may be present in some batches of EDTA). The cleavage of the DNA is site-specific and the average fragment size resulting from limit digestion of total S. lividans DNA is about 6kb. DNA from Streptomyces coelicolor A3(2) and several other Streptomyces strains, and from E. coli, is not cleaved under the same conditions. A S. lividans mutant has been isolated which lacks the DNA modification. We suspect that many reports of "poor" preparations of S. lividans plasmids may be due to the above effect. 相似文献
17.
A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae 总被引:55,自引:0,他引:55
We have detected two site-specific endonucleases in strains of Saccharomyces cerevisiae. One endonuclease, which we call YZ endo, is present only in yeast strains that are undergoing mating-type interconversion. The site at which YZ endo cleaves corresponds to the in vivo double-strand break occurring at the mating-type locus in yeast undergoing mating-type interconversion. YZ endo generates a site-specific double-strand break having 4-base 3' extensions terminating in 3' hydroxyl groups. The site of cleavage occurs in the Z1 region near the YZ junction of the mating-type locus. Mutant mating-type loci known to decrease the frequency of mating-type interconversion are correspondingly poor substrates for YZ endo in vitro. In vitro analysis of a number of such altered recognition sites has delimited the sequences required for cleavage. The molecular genetics of mating-type interconversion is discussed in the context of this endonucleolytic activity. The second endonuclease, which we refer to as Sce II, is present in all strains of S. cerevisiae we have examined. The cleavage site of Sce II has been determined and proves to be unrelated to the cleavage site of YZ endo. 相似文献
18.
Goodarzi AA Yu Y Riballo E Douglas P Walker SA Ye R Härer C Marchetti C Morrice N Jeggo PA Lees-Miller SP 《The EMBO journal》2006,25(16):3880-3889
The Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double-strand breaks (DSBs) in an ATM and DNA-PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA-PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation at the T2609-T2647 cluster, in the presence of Ku and target DNA, is required for Artemis-mediated endonuclease activity. Moreover, autophosphorylated DNA-PKcs stably associates with Ku-bound DNA with large single-stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA-PK that enhance Artemis cleavage at single-strand to double-strand DNA junctions. These findings demonstrate that DNA-PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing. 相似文献
19.
20.
Poly(ADP-RIBOSE) polymerase-1 (Parp-1) antagonizes topoisomerase I-dependent recombination stimulation by P53 总被引:2,自引:1,他引:1
PARP-1 interacts with and poly(ADP-ribosyl)ates p53 and topoisomerase I, which both participate in DNA recombination. Previously, we showed that PARP-1 downregulates homology-directed double-strand break (DSB) repair. We also discovered that, despite the well-established role of p53 as a global suppressor of error-prone recombination, p53 enhances homologous recombination (HR) at the RARα breakpoint cluster region (bcr) comprising topoisomerase I recognition sites. Using an SV40-based assay and isogenic cell lines differing in the p53 and PARP-1 status we demonstrate that PARP-1 counteracts HR enhancement by p53, although DNA replication was largely unaffected. When the same DNA element was integrated in an episomal recombination plasmid, both p53 and PARP-1 exerted anti-recombinogenic rather than stimulatory activities. Strikingly, with DNA substrates integrated into cellular chromosomes, enhancement of HR by p53 and antagonistic PARP-1 action was seen, very similar to the HR of viral minichromosomes. siRNA-mediated knockdown revealed the essential role of topoisomerase I in this regulatory mechanism. However, after I-SceI-meganuclease-mediated cleavage of the chromosomally integrated substrate, no topoisomerase I-dependent effects by p53 and PARP-1 were observed. Our data further indicate that PARP-1, probably through topoisomerase I interactions rather than poly(ADP-ribosyl)ation, prevents p53 from stimulating spontaneous HR on chromosomes via topoisomerase I activity. 相似文献