Nuclease S1-sensitive sites on superhelical DNA molecules carrying the LTR region of Moloney murine leukemia virus

The long terminal repeat (LTR) from proviral DNA of Moloney murine leukemia virus (Mo-MLV) was cloned on a derivative of pBR322, and after introducing superhelical torsions into the resulting recombinant, the sites of conformational transition were investigated by the nuclease S1-digestion method. With an increase in the negative linking differences, fourteen dominant cutting sites were identified, of which two were mapped inside the LTR and one at the 3' end of the LTR. By searching the sequence data, all these sites were localized in the regions having either palindromic sequences or AT-rich sequences. Free energy calculation for the local secondary structure on one strand indicated that nuclease S1 attacked the palindromic sequence regions which could form relatively stable hairpin structures. Under the conditions used, no correlation was found between the S1-sensitive sites and the potential Z-DNA-forming regions, including those within the enhancer sequence.     

S1 sensitive sites in adenovirus DNA.

S1 nuclease has been used as a probe for regions of DNA secondary structure in supercoiled recombinant plasmids containing adenovirus (Ad) DNA sequences. In the sequences examined two S1 sensitive sites were identified in the left-terminal 16.5% of Ad 12 DNA, one of which aligned approximately with an inverted repeat region in the DNA sequence. In addition an S1 sensitive site was dictated by a potential cruciform structure in the region of the Ad 2 major late promoter. In contrast to the expected cleavage site at the loop of the cruciform, cleavage occurred at the base of the stem in the region of the TATA box. All three S1 sensitive sites identified were more sensitive to S1 than the endogenous sites in the parent plasmids.     

Changes in wheat DNA methylation pattern after chronic seed gamma-irradiation

Alterations of DNA methylation patterns of wheat two varieties--Odessa' albatross and Donetsk 48 have been studied. Seeds were irradiated for 4 months with low dose rate (3 x 10(-7) Gy/s). Six restriction endonucleases were used in the experiments. Primary distinction in DNA methylation patterns of the studied varieties has been demonstrated. The chronic irradiation resulted in the increase of methylation level on the sites of recognition for Glul and Sou3Al and in the decline of this index for the sites of recognition of GlaI and HpaII. The meaningful increase of chromosome aberration levels was demonstrated at the same accumulated dose of chronic irradiation. The role of changes of DNA methylation patterns in development of radiation damage and organism protective reactions is discussed.     

Unpaired bases in phage DNA after gamma-irradiation in-situ and in-vitro

Summary Phage Lambda DNA, gamma-irradiated in-situ and in-vitro, has been analyzed for unpaired bases by melting, reannealing, and cleavage with Sl nuclease which is specific for single-stranded DNA. DNA, irradiated in-situ, i.e., in the phage particle, contained sites being sensitive to Sl nuclease. These single-stranded lesions were passed over and conserved during reannealing, whereas adjacent DNA regions reannealed specifically. Complementary base-pairing was restored after Sl nuclease treatment. Comparison of the T_m,-data before and after Sl nuclease treatment indicated that the single-stranded regions were removed by the enzyme. In contrast, DNA irradiated in-vitro, i.e., gamma-irradiated in aqueous solution, failed to match complementarily and was not sensitive to Sl nuclease. Thus it appears that lesions leading to unpaired bases were randomly distributed in DNA irradiated in-vitro, but occurred in clusters after irradiation in-situ. Most probably these clusters contain damaged bases which in turn caused localized disruption of the hydrogen bonds between complementary base pairs.     

Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage.

DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.     

Atmnd1-delta1 is sensitive to gamma-irradiation and defective in meiotic DNA repair

The efficient repair of double-strand breaks (DSBs) in genomic DNA is crucial for the survival of all organisms. Mnd1 is suggested to promote the strand invasion step during meiotic recombination. We used a forward genetics approach, through the search for mutants, to characterize the Arabidopsis homologue of Mnd1. Atmnd1 null mutants exhibit normal vegetative and flower development. In contrast, during prophase I, chromosomes become fragmented resulting in random distribution of the fragments between polyads. Male and female meiosis are defective and strong accumulation of AtRAD51 was observed in atmnd1-delta1 nuclei. These results suggest that similarly to its yeast and animal homologues, AtMnd1 plays a role in DSB repair during meiosis. In addition, like its human homologue AtMnd1 is expressed in somatic cells. AtMnd1 expression is strongly induced by gamma-rays and null mutants show increased sensibility to gamma-rays. Taken together, these results suggest that AtMnd1 is also required for DSB repair in somatic cells.     

Changes in the kinetics of rabbit thymus DNA re-association in various time periods after gamma-irradiation

A distinction has been detected in the kinetic curves of the control and in vivo irradiated DNA at the site of the reassociation of moderate and unique genome fractions. This distinction increased with increasing Cot. It was shown that the thermostability decreased and the profiles of DNA duplex thermoelution changed in irradiated rabbits as compared with intact ones.     

Production of dihydrothymidine stereoisomers in DNA by gamma-irradiation

5,6-Dihydrothymidine (dDHT) is a derivative thymidine formed during gamma-irradiation. This paper demonstrates the conditions under which dDHT is formed in solutions of DNA and that dDHT is produced in the DNA of HeLa cells during gamma-irradiation. The product of dDHT by gamma-irradiation of either thymidine or DNA has been quantitated by a sensitive and specific high-pressure liquid chromatography method. dDHT is a major product of the anoxic irradiation of thymidine (G value 0.5) but is produced in substantially smaller amounts in DNA irradiated under the same conditions (G value 0.026). The presence of oxygen reduces the yield of dDHT by at least 25-fold for both irradiation substrates. In HeLa cells, 60Co irradiation under anoxia produces (6.2 +/- 0.2) X 10(-8) mol of the R isomer of dDHT per mole of cell deoxynucleotide per gray (G value 0.11). gamma-Irradiation of thymidine produces equal quantities of the R and S stereoisomers of dDHT. Irradiation of DNA produces significantly more (69%) (R)- than (S)-dDHT. DNA isolated from cultured human cells following gamma-irradiation also contains more of the R than the S form of dDHT. The conformation of double-stranded DNA favors a stereospecific production of the R isomer. Among products of gamma-irradiation of DNA, dDHT is unique in its strict requirement for anoxia during irradiation and the preferential production of a particular stereoisomer.     

Phosphorylation and dephosphorylation of calsequestrin on CK2-sensitive sites in heart

Calsequestrin (CSQ) concentrates in junctional sarcoplasmic reticulum (SR) where it functions in regulation of Ca2+ release. When purified from heart tissue, cardiac CSQ contains phosphate on a cluster of C-terminal serine residues, but little is known about the cellular site of kinase action, and the identity of the kinase remains uncertain. To determine basic features of the phosphorylation, we examined the reaction in canine heart preparations. CSQ phosphorylation was observed in [32P]metabolically-labeled heart cells after adenoviral overexpression, and its constitutive phosphorylation was limited to a CK2-sensitive C-terminal serine cluster. The CSQ kinase was oriented intralumenally, as was CSQ, inside membrane vesicles, such that exposure to each required detergent permeabilization. Yet even after detergent permeabilization, CSQ was phosphorylated much less efficiently by protein kinase CK2 in cardiac microsomes than was purified CSQ. Reduced phosphorylation was strongly dependent upon protein concentration, and phosphorylation time courses revealed a phosphatase activity that occurred constitutively as phosphorylated substrate accumulates. Evidence of selective dephosphorylation of CSQ glycoforms in heart homogenates was also seen by mass spectrometry analysis. Molecules with greater mannose content, a feature of early secretory pathway compartments, were more highly phosphorylated, while greater dephosphorylation was apparent in more distal compartments. Taken together, the analyses of CSQ phosphorylation in heart suggest that a constitutive process of phosphate turnover occurs for cardiac CSQ perhaps associated with its intracellular transport.     

pH changes in Chinese hamster cells after gamma-irradiation

Intracellular pH (pHin) changes after gamma-irradiation of Chinese hamster fibroblasts have been studied by a fluorescence method using the ratio of fluorescence intensities after excitation at 488 and 458 nm and measurement at emission wavelength of 515 nm. Irradiation with doses inducing reproductive death (2.5-20 Gy) causes a pHin shift towards the alkaline region by 0.4-0.5 pH units, but this shift is transient. Irradiation with a 500 Gy dose, inducing interphase death, causes a more pronounced (pHin greater than or equal to 8.0) alkalization of the intracellular medium which is retained for more than 1.5 hours post-irradiation. It is proposed that the observed alkalization of the internal medium of irradiated cells is possibly due to a change in the functional state of mitochondria. These changes are probably one of the causes of interphase cell death after irradiation with high doses.     

Structural changes in brucella chemical vaccine after gamma-irradiation

It was shown that gamma-irradiation of Brucella strain chemical vaccine stimulated phospholipid peroxidation therein: the content of extractable total phospholipids in the exposed vaccine decreased mainly due to diminution of phosphatidylcholines and phosphatidyl-ethanolamines. A relative content of high- and low-molecular weight protein components increased in the gamma-irradiated vaccine.     

DNA repair and antioxidant defence in the repair-deficient human cells (Marfan's syndrome) after gamma-irradiation

DNA repair synthesis (RS) was investigated in lymphocytes of healty donors and repair-deficient cells (Marfan's syndrome), treated with inhibitor of superoxidedismutase (SOD)--TRIEN--after gamma-irradiation. Significant difference was revealed in cells of healthy donors and Marfan's syndrome: in cells of healthy donors TRIEN stimulated DNA RS whereas this effect didn't observed in Marfan's syndrome cells. So it is possible to suppose that SOD activity is different in normal and gamma-repair-deficient cells.     

Meiotic recombination remote from prominent DNA break sites in S. pombe

DNA breakage is intimately associated with meiotic recombination in the fission yeast Schizosaccharomyces pombe. Sites of prominent DNA breakage were found approximately 25 to approximately 200 kb apart in the genomic regions surveyed. We examined in detail a 501 kb region of chromosome I and found six sites, or tight clusters of sites, at which approximately 2%-11% of the DNA accumulated breaks in a rad50S mutant. In contrast to the discrete, widely spaced distribution of prominent break sites, recombination in this region was more uniformly distributed (0.7-1.6 cM/10 kb) whether the genetic interval tested contained no, one, or more such sites. We infer that although recombination depends upon DNA breakage, recombination often occurs remote from these sites (tens of kilobases away); we discuss mechanisms by which this may occur.