Alkali-labile sites and post-irradiation effects in gamma-irradiated biologically active double-stranded DNA in aqueous solution.

Gamma-irradiation of double-stranded RF-DNA of bacteriophage phi X174 in aqueous solution in the presence of oxygen produces at least one type of alkali-labile site. It is lethal and gives rise to breaks by alkali and is identical with the damage which becomes manifest by post-irradiation heat treatment. The effect of alkali is dependent on temperature. Furthermore, the excision repair system is not involved in eliminating lethal nucleotide damage in RFI-DNA.     

Alkali-labile sites in daughter DNA of damagedEscherichia coli do not reflect apyrimidinic sites

Apyrimidinic sites arising after excision of uracil incorporated into DNA daughter strands might constitute alkalilabile sites. The hypothesis was checked in the present paper and the data obtained do not support it.     

Nanomechanical behaviors of microcantilever-based single-stranded DNA chips induced by counterion osmotic effects

Experiments show that deflections of microcantilever-DNA chip can be induced by many factors, such as grafting density, hybridization efficiency, concentration, length and sequence of DNA molecules, buffer salt concentration, time, and temperature variation. However, there are few theoretical works on microcantilever-DNA chips. The present paper is aimed to study the influence of counterion effects of single-stranded DNA (ssDNA) polyelectrolyte solution on the nanomechanical behaviors of microcantilever-based ssDNA chips during packing process. First, the effect of osmotic pressure induced by ingress of counterions into DNA brush structures is studied with Hagan’s model for a cylindrical polyelectrolyte brush system on the basis of Poisson-Boltzmann distribution hypothesis. Second, Zhang’s two-variable method for a laminated cantilever is used to formulate a four-layer energy model for ssDNA chips with weak interactions. Third, the influence of grafting density, ssDNA chain length, and salt concentration on packing deflection is investigated using the principle of minimum energy. The predictive tendency is qualitatively similar to those observed in some related ssDNA chip experiments. The difference between the four-layer model and the simplified two-layer model for ssDNA chips is also discussed.     

Isolation of specific ribosome binding sites from single-stranded DNA.

The ability of Escherichia coli ribosomes to protect small specific regions of single-stranded bacteriophage DNA from digestion by pancreatic DNAase has been investigated. A procedure is described by which ribosome-protected fragments can be isolated from the DNA of bacteriophage f1 and φX174. Size determination by polyacrylamide gel electrophoresis or thin layer homochromatography together with fingerprinting analysis following chemical depurination or digestion with E. coli endonuclease IV were employed to show that these fragments represent a small specific portion of these DNAs. The protection reaction is largely dependent upon components necessary for ribosome binding to mRNA, including GTP, formylmethionyl-tRNA, and initiation factors. Thus, ribosomal binding to DNA mimics the ribosome-mRNA interaction. Furthermore, the regions in f1 and φX174 DNA which are protected differ in sequence from each other.When E. coli endonuclease IV is substituted for pancreatic DNAase in the ribosome protection reaction, a fragment of φX174 DNA is obtained about 150 bases in length which contains all of the pyrimidine tracts in the shorter 50-base fragment obtained with pancreatic DNAase, and a number of additional polypyrimidines.Double-stranded DNAs such as φX174 replicative form do not bind at all to ribosomes in their native state. Heat denaturation of such double-stranded DNAs allows ribosome binding. Protection of the same specific regions as those protected in single-stranded φX174 DNA was observed. A similar specific protection was observed following heat denaturation and ribosome binding with DNA from polyoma virus.     

Termination sites of the in vitro DNA synthesis on single-stranded DNA photosensitized by promazines

Bacteriophage phi X174 and M13 mp9 single-stranded DNA molecules were primed either with restriction fragments or synthetic primers and irradiated with near UV light in the presence of promazine derivatives. These DNAs were used as template for in vitro complementary chain synthesis by Escherichia coli DNA polymerase I large fragment. Chain terminations were observed by denaturing polyacrylamide gel electrophoresis of the synthesis products and localized by comparison with a standard dideoxy sequencing pattern. More than 90% of the chain terminations were mapped exactly one nucleotide before a guanine residue. In addition, photoreaction was shown to occur more predominantly with guanine residues localized in single-stranded parts of the genome. The same guanine residues could also be damaged when the reaction was performed, in the dark, in the presence of the artificially generated promazine cation radicals. Using the BamHI-SmaI adaptor (5'GATCCCCGGG-3'), it was shown that the guanine alteration was a covalent addition of the promazine, or of a cation radical photodegradation product, on the guanine moiety. Kinetics of chlorpromazine photoaddition on single-stranded and double-stranded DNAs were determined.     

Repair of DNA damage induced by oxygen radicals in human non-proliferating and proliferating lymphocytes.

Repair of DNA lesions induced by oxygen radicals, generated by xanthine/xanthine oxidase (X/XO), was studied in human peripheral blood lymphocytes and in PHA-stimulated proliferating lymphocytes from 4 healthy subjects. The lesions included DNA-strand breaks (SSB) and other lesions that are converted to SSB under alkaline conditions. The frequencies of SSB were estimated by fluorometric analysis of DNA unwinding. Maximum production of SSB occurred within 10 min of incubation with X/XO at 22 degrees C; with 0.5 mM or higher concentrations of xanthine; and with 0.1-0.5 units/ml of xanthine oxidase. Proliferating lymphocytes repaired X/XO-induced SSB about 4 times more rapidly than lymphocytes. Lymphocytes repaired X/XO-induced SSB more slowly than SSB caused by gamma-radiation. These findings are consistent with the evidence that a number of DNA-repair enzymes have greater activity in proliferating cells than in resting cells. These findings also support the view that there are differences between the DNA damage due to oxygen radicals and that due to ionizing radiation.     

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.     

Transformation of Bacillus subtilis by single-stranded plasmid DNA.

The single-stranded form of a pE194-based plasmid transformed Bacillus subtilis protoplasts at least as efficiently as did the double-stranded plasmid, but the single-stranded form did not detectably transform B. subtilis competent cells.     

Strong binding of single-stranded DNA by stem-loop oligonucleotides.

We report that oligodeoxynucleotides which form stem-loop hairpin structures and which have pyrimidine-rich loops can form strong complexes with complementary single-stranded DNA sequences. Stem-loop oligonucleotides were constructed with a 25-nt T-rich loop and with variable Watson-Crick stems. The complexes of these oligomers with the sequence dA8 were studied by thermal denaturation. Evidence is presented that the complexes are one-to-one, bimolecular complexes in which the pyrimidine loop bases comprise the outer strands in a pyr.pur.pyr triplex, in effect chelating the purine strand in the center of the loop. Melting temperatures for the loop complexes are shown to be up to 29 degrees C higher than Watson-Crick duplex of the same length. It is shown that the presence of a stem increases stability of the triplex relative to an analogous oligomer without a stem. The effect of stem length on the stability of such a complex is examined. Such hairpin oligomers represent a new approach to the sequence-specific binding of single-stranded RNA and DNA. In addition, the finding raises the possibility that such a complex may exist in natural RNA folded sequences.     

Different base per unit length ratios exist in single-stranded RNA and single-stranded DNA.

A significant difference was found to exist in the number of bases per unit length of single-stranded RNA as compared to single-stranded DNA when single-stranded RNA or DNA molecules of known nucleotide sequence were measured by electron microscopy using a cytochrome spreading technique. Using this technique, single-stranded RNA was found to have 17.5% more bases per unit of length than single-stranded DNA. These ratios were verified using four different denaturing conditions for the RNA: 80% formamide, 80% formamide plus glyoxal, 80% formamide/4M urea and 80% formamide/4M urea plus glyoxal. Molecules ranging in size from 1541 to 5386 nucleotides were examined and the number of bases per unit length was found to vary inversely with micrometer was consistent when RNA and DNA molecules of the same length were compared.     

Photochemically induced RNA and DNA abasic sites

Two phosphoramidite building blocks were synthesized that can easily be deprotected by UV light to reveal natural abasic sites in oligoribonucleotides as well as in oligodeoxyribonucleotides. Another building block which releases a 2'-O-methylated abasic site upon UV radiation is also described.     

Sequence specificity of heat-labile sites in DNA induced by mitomycin C

The sequence specificity of the mitomycin C-DNA interaction was directly determined by using DNA sequencing techniques and by using 3'- or 5'-end-labeled DNA fragments of defined sequence as substrates. Mitomycin C reduced with sodium borohydride induced heat-labile sites in DNA preferentially at specific sequences. The heat-labile sites were induced most preferentially at the dinucleotide sequence G-T ( especially Pu G-T), which was determined by scanning autoradiograms with a microdensitometer after gel electrophoresis. DNA was cleaved at the 3' side of deoxyguanosines and of some deoxyadenosines by heat treatment. Oligonucleotides produced by heat treatment after reaction with reduced mitomycin C contained phosphoryl groups at the 5' termini. The 3' termini seemed not to have simple structures, judging from their electrophoretic mobilities. Oxygen radicals such as singlet oxygen and hydroxyl radical were possibly involved in the induction of heat-labile sites.     

Alkali-labile colicinogenic factor E1 DNA molecules formed in the presence of N-methyl-N'-nitro-N-nitrosoguanidine.

Plasmid ColE1 DNA of E. coli was used as a target DNA molecule to analyse the structural modification of DNA by N-methyl-N′-nitro-N-nitrosoguanidine. When the low concentration of this drug was used, both cell growth and overall DNA synthesis were neither stimulated nor inhibited, and plasmid DNA molecules were isolated as closed circles after replication. These molecules were stable for the ribonuclease treatment, but became susceptible to the alkaline hydrolysis. Such alkali-labile sites of ColE1 DNA were found in the parental strands and randomly distributed from the restriction endonuclease EcoR1 cleavage site.     

Chlamydia pneumoniae AP endonuclease IV could cleave AP sites of double- and single-stranded DNA

Endonuclease IV gene, the only putative AP endonuclease of C. pneumoniae genome, was cloned into pET28a. Recombinant C. pneumoniae endonuclease I V (CpEndoIV) was expressed in E. coli and purified to homogeneity. CpEndoIV has endonuclease activity against apurinic/apyrimidinic sites (AP sites) of double-stranded (ds) oligonucleotides. AP endonuclease activity of CpEndoIV was promoted by divalent metal ions Mg2+ and Zn2+, and inhibited by EDTA. The natural (A, T, C and G) and modified (U, I and 8-oxo-G (GO)) bases opposite AP site had little effect on the cleavage efficiency of AP site of ds oligonucleotides by CpEndoIV. However, the CpEndoIV-dependent cleavage of AP site opposite modified base GO was strongly inhibited by Chlamydia DNA glycosylase MutY. Interestingly, the AP site in single-stranded (ss) oligonucleotides was also the effective substrate of CpEndoIV. Similar to E. coli endonuclease IV, AP endonuclease activity of CpEndoIV was also heat-stable to some extent, with a half time of 5 min at 60 degrees C.     

High-molecular-weight linear multimer formation by single-stranded DNA plasmids in Escherichia coli.

We inserted foreign DNA segments into plasmids which replicate by a rolling-circle mechanism in Escherichia coli and observed the appearance of high-molecular-weight plasmid multimers (HMW). This phenomenon, which occurs more frequently with GC-rich segments, depends on the mode of replication of the plasmid and on host homologous recombination functions. We found that (i) HMW are formed upon insertion of a foreign DNA segment into a single-stranded DNA plasmid, whereas the same DNA insert has no such effect on a theta replicon, and (ii) HMW are not present in a recA mutant strain but are found in a lexA (Ind-) mutant. Enzymatic studies allowed us to define the HMW structure as linear double-stranded tandem head-to-tail plasmid repeats. Use of heteroplasmid strains showed that HMW production by one plasmid does not affect another resident plasmid, indicating that no host functions are phenotypically inactivated. This distinguishes our system from the HMW observed with various replicons in the absence of RecBCD enzyme activity. We propose that the role of the foreign insert is to protect the DNA from RecBCD exonuclease attack.