Chromosomal DNA Breaks Research Articles

A Gyrase Mutant with Low Activity Disrupts Supercoiling at the Replication Terminus

When a mutation in an essential gene shows a temperature-sensitive phenotype, one usually assumes that the protein is inactive at nonpermissive temperature. DNA gyrase is an essential bacterial enzyme composed of two subunits, GyrA and GyrB. The gyrB652 mutation results from a single base change that substitutes a serine residue for arginine 436 (R436-S) in the GyrB protein. At 42 degrees C, strains with the gyrB652 allele stop DNA replication, and at 37 degrees C, such strains grow but have RecA-dependent SOS induction and show constitutive RecBCD-dependent DNA degradation. Surprisingly, the GyrB652 protein is not inactive at 42 degrees C in vivo or in vitro and it doesn't directly produce breaks in chromosomal DNA. Rather, this mutant has a low k(cat) compared to wild-type GyrB subunit. With more than twice the normal mean number of supercoil domains, this gyrase hypomorph is prone to fork collapse and topological chaos near the terminus of DNA replication.

Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases.

Bloom's syndrome (BS), a disorder associated with genomic instability and cancer predisposition, results from defects in the Bloom's helicase (BLM) protein. In BS cells, chromosomal abnormalities such as sister chromatid exchanges occur at highly elevated rates. Using Xenopus egg extracts, we have studied Xenopus BLM (Xblm) during both unperturbed and disrupted DNA replication cycles. Xblm binds to replicating chromatin and becomes highly phosphorylated in the presence of DNA replication blocks. This phosphorylation depends on Xenopus ATR (Xatr) and Xenopus Rad17 (Xrad17), but not Claspin. Xblm and Xenopus topoisomerase IIIα (Xtop3α) interact in a regulated manner and associate with replicating chromatin interdependently. Immunodepletion of Xblm from egg extracts results in accumulation of chromosomal DNA breaks during both normal and perturbed DNA replication cycles. Disruption of the interaction between Xblm and Xtop3α has similar effects. The occurrence of DNA damage in the absence of Xblm, even without any exogenous insult to the DNA, may help to explain the genesis of chromosomal defects in BS cells.

Using nucleases to stimulate homologous recombination.

In essentially all organisms, double-strand breaks in chromosomal DNA stimulate repair by multiple mechanisms, including homologous recombination. It is possible to use site-specific reagents to produce a break or other recombinagenic damage at a unique site, which makes possible detailed analysis of the repair products. In addition, targeted mutagenesis and gene replacement are stimulated in the immediate vicinity of the break site. To utilize meganucleases with long recognition sequences, it is necessary to introduce the corresponding sequence prior to directed cleavage. The same is typically true of triplex-forming oligonucleotides that target polypurine-polypyrimidine tracts. Zinc-finger nucleases have the potential of being targetable to arbitrarily selected sites, owing to the flexibility of zinc finger recognition of DNA.

DNA repair: Rad52 – the means to an end

In eukaryotic organisms, double-strand breaks in chromosomal DNA are repaired either by non-homologous end-joining, or by homologous recombination. How do cells choose which pathway to use?

CpG islands detected by self-primed in situ labeling (SPRINS).

We have studied the distribution and methylation of CpG islands on human chromosomes, using the novel technique of self-primed in situ labeling (SPRINS). The SPRINS technique is a hybrid of the two techniques primed in situ labeling (PRINS) and nick translation in situ. SPRINS detects chromosomal DNA breaks, as in nick translation in situ, and not annealed primers, as is the case in PRINS. We analyzed in situ-generated DNA breaks induced by the restriction enzymes HpaII and MspI. These restriction enzymes enable the detection of chromosomal CpG islands. Both HpaII- and MspI-SPRINS produce a banding pattern resembling R-banding, indicating a higher level of CpG islands in R-positive bands than in R-negative bands. Our SPRINS banding observations also indicate differences in sequence copy number in the satellites of homologous acrocentric chromosomes. Furthermore, a comparison of homologous HpaII-SPRINS-banded X chromosomes of females from lymphocyte cultures grown without methotrexate or bromodeoxyuridine revealed methylation difference between them. The same comparison of homologous X chromosomes from the cell line GM01202D, which has four X chromosomes, one active and three inactive, revealed the active X chromosome to be hypermethylated.

Participation of the human p53 3'UTR in translational repression and activation following gamma-irradiation.

p53 protein levels have been shown to increase in a number of cells after treatment with genotoxic agents through a post-transcriptional mechanism. In gamma-irradiated human cells, the accumulation of p53 protein is accompanied by an increase in the association of p53 mRNA with large polysomes without any change in the level of p53 mRNA. This redistribution of p53 mRNA on polysomes in response to irradiation is consistent with enhanced translational activity of p53 mRNA. We demonstrate that a region of the p53 3'-untranslated region (3'UTR) inhibits translation of a chimeric reporter mRNA in vivo. Induced elevation of reporter activity after gamma-irradiation was seen in cells expressing chimeric reporter-p53 3'UTR transcripts. These data taken together demonstrate translational control of p53 gene expression after gamma-irradiation and denote a previously unsuspected and novel role for the p53 3'UTR in controlling translation.

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.

A novel nuclease activity from Xenopus laevis releases short oligomers from 5'-ends of double- and single-stranded DNA.

Double-strand breaks in chromosomal DNA of eucaryotic cells are assumed to be repaired by mechanisms of illegitimate recombination capable of direct rejoining of the broken ends. Cell-free extracts of Xenopus laevis eggs efficiently perform these end joining reactions with any pair of noncomplementary DNA termini whose single-stranded 5'- or 3'-overhangs do not exceed a length of approximately 10 nt. Using hairpin-shaped oligonucleotides that allow the construction of double-strand break termini with 5'- or 3'-overhangs of defined length and sequence we show that 5'-overhangs of more than 9-10 nt are exonucleolytically resected in the extract to produce shorter 5'-overhangs that can be metabolized in the end joining reaction. 5'-recessed ends in double-stranded DNA with 3'-overhangs of more than 2nt as well as the 5'-ends of single-stranded DNA also serve as substrates for the exonuclease activity. In all cases, oligomers of about 10 nt are released from the 5'-ends. We describe here a novel 5'-exonuclease activity present in eggs from Xenopus laevis that reproducibly removes decameric oligonucleotides from 5'-ends of double- and single-stranded DNA. A possible function of this unusual activity is discussed in the context of homologous and illegitimate genetic recombination processes.

Apoptosis of Retinal Ganglion Cells in Anterior Ischemic Optic Neuropathy

We identified retinal ganglion cells undergoing apoptosis, a form of programmed cell death, in an eye of a 70-year-old man with anterior ischemic optic neuropathy. The TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin end nick labeling) staining and the presence of condensed, fragmented nuclear bodies were used to identify apoptotic cells. Examination of TUNEL-stained retinal sections revealed occasional cells in the ganglion cell layer with pyknotic nuclei and brown reaction product, representing positive staining for chromosomal DNA breaks. Positive cells were sparsely distributed, consistent with the limited time in which apoptotic cells are identifiable before they are removed. The most likely explanation for these results is that injury to the retinal ganglion cell axon induces apoptosis. To our knowledge, this is the first report of human retinal ganglion cell apoptosis in an acute optic neuropathy.

Meiotic recombination break points resolve at high rates at the 5' end of a maize coding sequence.

Sequence analysis of recombination break points has defined a 377-bp recombination hot spot within the anthocyanin 1 (a1) gene. One-fifth of all recombination events that occurred within the 140-kb a1-shrunken 2 interval resolved within this 377-bp hot spot. In yeast, meiotic double-strand breaks in chromosomal DNA are thought to initiate recombination and are generally located 5' of coding regions, near transcription promoter sequences. Because the a1 recombination hot spot is located within the 5' transcribed region of the a1 gene, the sites at which recombination events initiate and resolve appear to be different, but both appear to be regulated in relation to transcribed sequences. Although transposon insertions are known to suppress recombination and alter the ratio of crossovers to apparent gene conversions, the Mutator 1 transposon insertion in the a1-mum2 allele does not alter the sites at which recombination events resolve.

Studies on the influence of cytosine methylation on DNA recombination and end-joining in mammalian cells.

To test the influence of cytosine methylation on homologous recombination and the rejoining of DNA double strand breaks in mammalian cells, we developed a sensitive and quantitative assay system using extrachromosomal substrates. First, methylation was introduced into substrates in vitro with the prokaryotic SssI methylase, which specifically methylates the C-5 position of cytosine bases within CpG dinucleotides, mimicking the mammalian DNA methyltransferase. Next, methylated substrates were incubated in mammalian cells for a sufficient length of time to recombine or rejoin prior to substrate recovery. Results from bacterial transformation of the substrates and from direct Southern analysis demonstrate that cytosine methylation has no detectable effect on either DNA end-joining or homologous recombination. Thus, the components of the protein machinery involved in these complex processes are unaffected by the major DNA modification in mammalian cells. These results leave open the possibility that methylation may modulate the accessibility of these components to chromosomal DNA by altering local chromatin structure.

Direct stimulation of poly(ADP ribose) polymerase in permeabilized cells by double-stranded DNA oligomers

Poly(ADP ribosyl)ation, a post-translational modification of nuclear proteins catalyzed by poly(ADP ribose) polymerase, is an immediate response of most eukaryotic cells to DNA strand breaks and has been implicated in DNA repair and other cellular phenomena associated with DNA strand breakage. Poly(ADP ribose) polymerase activity levels have been frequently assayed by incubating permeabilized cells with radioactively labeled NAD + as substrate. In such assays enzyme activation has routinely been achieved indirectly by prior exposure of living cells to carcinogens or by adding DNase I to permeabilized cells, thereby introducing strand breaks in chromosomal DNA. Here we show that, as an alternative method, the direct activation of purified poly(ADP ribose) polymerase by double-stranded oligonucleotides (N. A. Berger and S. I. Petzold, 1985, Biochemistry 24, 4325–4355) can be adopted for permeabilized cell systems. The inclusion of a palindromic decameric deoxynucleotide in the reaction buffer stimulated the enzyme activity in permeabilized Molt-3 human lymphoma cells up to 30-fold (at 5 μg/ml oligonucleotide concentration) in a concentration-dependent manner. The activating effect of oligonucleotides was also evident when ethanol-fixed HeLa cells were postincubated with NAD + to allow poly(ADP ribose) synthesis to occur in situ, which was detected as specific anti-poly(ADP ribose) immunofluorescence. We conclude that double-stranded oligonucleotides can be conveniently used as chemically and stoichiometrically well-defined poly(ADP ribose) polymerase activators in permeabilized or ethanol-fixed mammalian cells.

P-element transposase induces male recombination inDrosophila melanogaster

Male recombination in P-M dysgenic crosses has been viewed as a reflection of P-element transposase interacting with P elements. However, recent studies suggest that the transposase may catalyse double-stranded breaks in chromosomal DNA. We have, therefore, introduced P(delta 2-3 ry+) (99B), a single non-mobile P-element transposase source, into the long-standing laboratory true M strains of a flanking lethal crossover selective system, thus facilitating the examination of rare male recombination events as an assay for transposase activity. We find that the rate of male recombination in the presence of this non-mobile P element is greater than twenty times the background rate of male recombination in the control examined prior to introduction of the transposase source.

A specialized form of chromosomal DNA degradation induced by thymidylate stress in mouse FM3A cells

Thymidylate synthase-negative mutant mouse cells starved of thymidine or their parental FM3A cells treated with 5-fluoro-2′-deoxyuridine produced DNA fragments ranging from 50 to 200 kilobase pairs with a peak at 100 kb in length as determined by pulsed-field agarose gel electrophoresis. Accumulation of the DNA fragments following such thymidylate stress was time-dependent but their size distribution did not change in either case. Regions of the chromosomal DNA breaks seemed to be restricted to those where DNA replication was in progress as shown by pulse-labeling of the DNA synthesis. Emetine, an inhibitor of protein synthesis, blocked the accumulation of the DNA fragments when present during thymidylate stress. Cell-free extracts prepared from the thymidylate-stressed cells derived by either of the above means were capable of degrading DNA in chromatins prepared from normally growing cells in vitro. The resulting DNA fragments were similar but with a somewhat broader size distribution compared to those produced in vivo. The broader distribution of the fragments produced in the in vitro reaction became closer to the pattern obtained in vivo when ATP and 4 deoxyribonucleotides were added to the reaction.

Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I.

Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug-induced cleavable complex.

The repair of double-strand breaks in chromosomal DNA of yeast.

Ionizing radiation induces double-strand breaks in the nuclear DNA of the yeast Saccharomyces cerevisiae with an efficiency of approximately 0.6 X 10(-10) breaks being repaired. Based on the efficiency of break production, the sensitivity of a rad52 mutant, and the absence of radiation-induced recombination in such a mutant, it is proposed that the corresponding gene product may be involved in double-strand break repair. A model involving recombination and DNA synthesis is described for this type of repair.