When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (gamma-H2AX) demonstrates that gamma-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of (125)I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of (125)IdU and processed immunocytochemically to determine the number of gamma-H2AX foci. The numbers of (125)IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of (125)I decays and the number of foci per cell, consistent with the assumptions that each (125)I decay yields a DNA DSB and each DNA DSB yields a visible gamma-H2AX focus. Based on these findings, we conclude that gamma-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.

We discuss the role of proteins in promoting the branch migration step during homologous recombination.Genetic recombination involving the exchange of genetic information between two DNA molecules has been observed in virtually all organisms. An important intermediate in both homologous and site-specific recombination is the Holliday junction, the branch point connecting two duplex DNAs that are undergoing recombination. If the branch point is flanked by DNA sequence homology, the Holliday junction can spontaneously migrate in either direction by the exchange of hydrogen bonds between the bases in homologous DNA strands. This process is known as branch migration and is an important step in genetic recombination. In homologous recombination, the extent of branch migration affects the amount of genetic information that is transferred between two homologues. In site-specific recombination, such as in the integration of bacteriophage A into the Escherichia coli chromosome, branch migration in the region of homology shared between donor and recipient sites is a prerequisite for subsequent steps in the recombination pathway. Early estimates of the rate of branch migration yielded a step rate of several thousand bases per sec (1), a rate that was robust enough to be accommodated within the time scale of recombination in bacteriophages. This suggested that branch migration could occur spontaneously during recombination without the need for proteins to facilitate this step. Later, several groups studying cruciform transitions in supercoiled DNA obtained data suggesting that the rate of spontaneous branch migration might be significantly slower (2-4).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2021In addition to knowing the inherent rate of branch migration, it is also critical to know whether spontaneous branch migration can traverse sequence heterology such as mismatches, insertions, and deletions since homologous recombination usually involves the exchange of DNA strands between two similar but not identical duplexes. We recently observed that a single base mismatch was sufficient to slow the overall rate of branch migration (5). Moreover, this attenuation by sequence heterology was more pronounced in magnesium than in sodium, suggesting that branch migration is influenced by metal ions.To clarify questions concerning the intrinsic rate of branch migration, we have developed an improved assay for branch migration to determine kinetic parameters of spontaneous DNA branch migration as a function of temperature and ionic conditions. We observe that branch migration in magnesium is '1000 times slower than was previously reported. In the absence of magnesium, branch migration is 3 orders of magnitude faster. We discuss the relationship between the structure of the Holliday junction and rates of branch migration and the need for proteins to promote branc...

That irradiated cells affect their unirradiated 'bystander' neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNArepair genes in the bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named c-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces c-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple c-H2AX foci. After 18 h coculture with cells irradiated with 20 a-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with c-rays, and in cultures containing media conditioned on c-irradiated cells. DNA DSB repair proteins accumulated at c-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying c-H2AX focus formation may be responsible for its downstream manifestations.

A repeated non-coding DNA sequence d(TTAGGG)n is present in the telomeric ends of all human chromosomes. These repeats can adopt multiple inter and intramolecular non-B-DNA conformations that may play an important role in biological processes. Two intramolecular structures of the telomeric oligonucleotide dAGGG(TTAGGG)3, antiparallel and parallel, have been solved by NMR and X-ray crystallography. In both structures, the telomeric sequence adopts an intramolecular quadruplex structure that is stabilized by G-4 quartets, but the ways in which the sequence folds into the quadruplex are different. The folds of the human telomeric DNA were described as an anti-parallel basket-type and a parallel propeller-type. We applied 125I-radioprobing to determine the conformation of the telomeric quadruplex in solution, in the presence of either Na+ or K+ ions. The probability of DNA breaks caused by decay of 125I is inversely related to the distance between the radionuclide and the sugar unit of the DNA backbone; hence, the conformation of the DNA backbone can be deduced from the distribution of breaks. The probability of breaks measured in the presence of Na+ and K+ were compared with the distances in basket-type and propeller-type quadruplexes obtained from the NMR and crystal structures. Our radioprobing data demonstrate that the antiparallel conformation was present in solution in the presence of both K+ and Na+. The preferable conformation in the Na+-containing solution was the basket-type antiparallel quadruplex whereas the presence of K+ favored the chair-type antiparallel quadruplex. Thus, we believe that the two antiparallel and the parallel conformations may coexist in solution, and that their relative proportion is determined by the type and concentration of ions.