DNA breakage drives nuclear search

The search for a homologous template is a fundamental, yet largely uncharacterized, reaction in DNA double-strand break repair. Two reports now demonstrate that broken chromosomes increase their movement and explore large volumes of nuclear space searching for a homologous template. Break mobility requires resection and recombination enzymes, as well as damage-checkpoint components.     

Modifying chromatin architecture during the response to DNA breakage

The human genome is compacted in a dynamic macromolecular complex, chromatin, whose structure presents a considerable barrier to the cellular machinery which responds to DNA double-strand breaks. This review discusses current understanding of the processes that modify chromatin architecture to enable, first, the sensing of DNA breakage, next, the assembly of the protein complexes that resolve the lesion, and finally, the restoration of epigenetic marks after its repair. The importance of these fundamental biological processes is underscored by the growing appreciation that they are aberrant in human diseases, and that their modulation could provide new approaches to disease therapy.     

Hydrodynamic shear breakage of DNA

The rate of breakage of duplex DNA molecules by laminar flow through a capillary has been studied. For λb2b5c DNA (molecular wt., M = 25 × 10⁶) the point at which breakage occurs is normally distributed around the center of the molecule with a standard deviation of 12.5% of the molecular length. At constant shear stress or shear rate, the breakage rate is independent of ionic strength. Thus, shear induced local denaturation is not a rate limiting, preliminary step in breakage. In experiments at constant temperature with varying solvent viscosity (controlled by added sucrose) the breakage rate is a function of shear rate, not of shear stress. The rate of opening of hydrogenbonded circles into linear molecules by hydrodynamic shear is also shown to be a function of shear rate and not of shear stress. The breakage rate at constant shear rate is not greatly dependent on temperature. The shear rate required to achieve breakage is inversely proportional to M^1,2. The breakage rate constant, k varies as a very high power of the shear rate; at 25°C, d In k/d In Gm ～ 15; at 10°C, d In k/d In Gm ～ 26, where Gm is the maximum shear rate at the capillary wall. The unexpected result that breakage rate is mainly dependent on shear rate, not shear stress, supports a model in which the DNA molecule is distorted with a driving force which depends on the hydrodynamic shear stress, ηG, but the rate limiting step is segment diffusion into a highly extended configuration. The characteristic time to achieve this configuration is proportional to solvent viscosity, η, hence the breakage rate is dependent on ηG/η or G, the shear rate.     

Shear breakage of DNA.

Determinations were made of the mean length of fragments produced after shearing long (greater than 100 kb) native Hela DNA in a VirTis homogenizer. (VirTis Co., Inc., Gardiner, N.Y.). The mean length (L) is a function of the speed of rotation of the homogenizer blades (omega), time of shearing (t), water concentration ([H2O]), solvent viscosity (eta), temperature (T), and energy of activation (E*), but not a function of the initial length so long as the starting molecules sustain an average of three or more breaks. The relationship of the parameters is expressed by the equation L = (b/omegat1/2eta1/2[H2O])eE*/2kBT, where kB is the Boltzmann constant and b is a constant of proportionality. The breakage rate constant k was determined to have the relationship k = (omega2L2eta[H2O]2/2b2)e-E*/kBT. These equations are valid throughout large ranges of the parameters, and a simple method is described which chooses a final mean length between at least 0.15 and 36 kb by choosing the appropriate shearing conditions and initial fragment length. The heterogeneity of shearing conditions within the shearing vessel permits use of the equations at all breakage rates tested. Based on the work of others using more homogeneous shearing conditions and initial fragment lengths, more complicated forms of the equations are necessary at low breakage rates but not at high ones. A proposed model of the breakage mechanism suggests that molecules with stress-induced localized denaturations break at a rate different from that for native DNA.     

Bleomycin-induced breakage of closed-circular DNA.

The amount of breakage induced by bleomycin in closed-circular DNA from bacteriophage PM2 was investigated under various reaction conditions. Analysis of the extent of breakage, by alkaline band centrifugation and agarose gel electrophoresis, yielded quite different results from those previously reported for the breakage of closed-circular SV40 DNA by bleomycin. Compared to these earlier results, we found (a) the reaction required the addition of 2-mercaptoethanol; and (b) the breakage reaction was further stimulated by the addition of 2-mercaptoethanol in the absence of EDTA.     

DNA strand breakage after kidney storage

Ischemia and storage cause single-stranded DNA breaks. The breaks become evident after 30 min of warm storage or after only 4 hr of cold storage. The number of breaks increases with increasing ischemia time. The DNA damage was detected by alkaline sucrose gradient analysis of the DNA.     

FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress

Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)–binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress.     

Strand breakage in DNA by silicic acid

The alkaline unwinding assay has been used to demonstrate the formation of single-strand breaks in DNA on treatment with silicic acid. Double-stranded DNA, containing no single-strand breaks, when incubated with increasing concentrations of silicic acid, showed the formation of an increasing number of strand breaks per molecule. Experiments on reduction of silicic acid-treated DNA with NaBH4 suggested the possibility of creation of apurinic or apyrimidinic sites. The significance of silicic acid interaction with cellular DNA during asbestos exposure is discussed.     

DNA gyrase complex with DNA: determinants for site-specific DNA breakage.

DNA gyrase catalyses DNA supercoiling by making a transient double-stranded DNA break within its 120-150 bp binding site on DNA. Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme-DNA intermediate inducing sequence-specific DNA cleavage and revealing potential sites of gyrase action on DNA. We have used site-directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322. Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro. Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes. The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies. The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage. These results are discussed in relation to the mechanism and structure of the gyrase complex.     

Single-strand breakage in DNA of Escherichia coli exposed to Cd2+.

When a growing culture of Escherichia coli was exposed to 3 X 10(-6) M Cd2+, 85 to 95% of the cells lost their ability to form colonies on agar plates. Loss of viability was accompanied by considerable single-strand breakage in the DNA, with no detectable increase in double-strand breaks. A direct correlation appeared to exist between the number of single-strand breaks and the concentrations of Cd2+ to which the cells were exposed. Exposure of DNA in vitro to a Cd2+ concentration of 3 X 10(-6) M or higher, followed by sedimentation in alkaline sucrose gradients, demonstrated no single-strand breaks. Cadmium-exposed cells recovered viability when incubated in Cd2+-free liquid medium containing 10 mM hydroxyurea. During the early period of recovery, there was a lag in the incorporation of labeled thymidine, but cellular DNA, at least in part, appeared to be repaired.     

Changes in DNA properties due to treatment with the pesticides malathion and DDVP

Summary Properties of calf thymus DNA were investigated after treatment with the pesticides malathion (0,0-dimethyl-S-(1,2-bis ethoxycarbonyl ethyl)dithiophosphate) and DDVP (0,0-dimethyl-0-(2,2 dichlorovinyl)phosphate) in vitro by means of derivative (differential) pulse polarography (DPP), thermal denaturation curves recorded spectrophotometrically (T_m), viscometric measurements, and chromatography on the hydroxyapatite column. Changes in the properties of DNA were observed by means of DPP after only a few hours incubation with the pesticides, whereas the other methods did not detect any changes even after 48 h. The results obtained by DPP indicate that single-stranded segments and thermolabile regions are formed in DNA due to the action of the pesticides. This behaviour could perhaps be a consequence of guanine alkylation followed by depurination and chain scission at elevated temperatures. Malathion and DDVP differ in the kinetics of reaction with double-helical DNA. DDVP is more reactive and its action is also manifested after 72 h in changes in viscosity, T_m, and chromatographic behaviour on the hydroxyapatite column. The changes induced by malathion were, under identical conditions, not detectable by these methods.     

Use of 'nuclear monolayers' to identify factors influencing DNA double-strand breakage by X-rays

Nuclear monolayers, prepared by treatment of mammalian cells with non-ionic detergents, showed increased sensitivity to X-ray-induced DNA double-strand breakage (dsb), as compared with intact cells, due to a decrease in the low-dose 'shoulder'. The DNA dsb dose-response shoulder could be restored by irradiating nuclei in the presence of sulphydryl compounds. However, the ineffectiveness of glutathione, when used at near cellular levels, in restoring the shoulder, suggested a possible role for protein sulphydryls in the radiation response of intact cells.     

Nijmegen breakage syndrome: clinical manifestation of defective response to DNA double-strand breaks

Nijmegen breakage syndrome is a rare autosomal recessive genetic disease belonging to a group of disorders often called chromosome instability syndromes. In addition to a characteristic facial appearance and microcephaly, patients suffering from Nijmegen breakage syndrome have a range of symptoms including radiosensitivity, immunodeficiency, increased cancer risk and growth retardation. The underlying gene, NBS1, is located on human chromosome 8q21 and codes for a protein product termed nibrin, Nbs1 or p95. Over 90% of patients are homozygous for a founder mutation: a deletion of five base pairs which leads to a framehift and protein truncation. The protein nibrin/Nbs1 is suspected to be involved in the cellular response to DNA damage caused by ionising irradiation, thus accounting for the radiosensitivity of Nijmegen breakage syndrome. We review here some of the more recent findings on the NBS1 gene and discuss how they impinge on the clinical manifestation of the disease.