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The mechanisms of gene amplification in tumour cells are poorly understood and the relationship between extrachromosomal DNA molecules, named double minutes (dmins), and intrachromosomal homogeneously staining regions (hsr) is not documented at nucleotide resolution. Using fluorescent in situ hybridization and whole genome sequencing, we studied a xenografted human oligodendroglioma where the co-amplification of the EGFR and MYC loci was present in the form of dmins at early passages and of an hsr at later passages. The amplified regions underwent multiple rearrangements and deletions during the formation of the dmins and their transformation into hsr. In both forms of amplification, non-homologous end-joining and microhomology-mediated end-joining rather than replication repair mechanisms prevailed in fusions. Small fragments, some of a few tens of base pairs, were associated in contigs. They came from clusters of breakpoints localized hundreds of kilobases apart in the amplified regions. The characteristics of some pairs of junctions suggest that at least some fragments were not fused randomly but could result from the concomitant repair of neighbouring breakpoints during the interaction of remote DNA sequences. This characterization at nucleotide resolution of the transition between extra- and intrachromosome amplifications highlights a hitherto uncharacterized organization of the amplified regions suggesting the involvement of new mechanisms in their formation., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The mechanisms of formation of intrachromosomal amplifications in tumours are still poorly understood. By using quantitative polymerase chain reaction, DNA sequencing, chromosome walking, in situ hybridization on metaphase chromosomes and whole-genome analysis, we studied two cancer cell lines containing an MYC oncogene amplification with acquired copies ectopically inserted in rearranged chromosomes 17. These intrachromosomal amplifications result from the integration of extrachromosomal DNA molecules. Replication stress could explain the formation of the double-strand breaks involved in their insertion and in the rearrangements of the targeted chromosomes. The sequences of the junctions indicate that homologous recombination was not involved in their formation and support a non-homologous end-joining process. The replication stress-inducible common fragile sites present in the amplicons may have driven the intrachromosomal amplifications. Mechanisms associating break-fusion-bridge cycles and/or chromosome fragmentation may have led to the formation of the uncovered complex structures. To our knowledge, this is the first characterization of an intrachromosomal amplification site at nucleotide resolution.

DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for 8 of the 9 studied genes (KU70, KU80, XRCC4, LIG4, Artemis, MRE11, RAD50, NBS1) but not for DNA-PKcs. No mutation was found in the remaining allele of the genes with LOH and the mRNA expression did not correlate with the allelic status. Our findings suggest that non-homologous end-joining repair pathway alteration is unlikely to be involved in the high genomic instability observed in these tumors.

Two modified polynucleotides having the Z-DNA conformation (poly [dG-dC] dien Pt and poly [dG-br5dC] . poly [dG-br5dC]) have been used for determination of antibodies to Z-DNA. Such antibodies were found in sera of patients with systemic lupus erythematosus and with Crohn's disease. They were scarcely observed in polyradiculoneuritis and in amyotrophic lateral sclerosis. In Crohn's disease sera, no antibodies to B-DNA were ever found but presence of two different families of antibodies to Z-DNA was demonstrated.

The study of poly(dI-br5dC).poly(dI-br5dC) films by infrared spectroscopy shows that in low salt concentration, the conformation of this polynucleotide belongs to the B-family and in high salt concentration to the Z-family. 31P nuclear magnetic resonance and circular dichroism confirm the existence of these two forms. By circular dichroism and ultraviolet absorption, it is shown that the equilibrium constant of the B reversible Z transition depends upon temperature. The deuteration rates of exchangeable protons involved in hydrogen bonds between base pairs were deduced from the changes in absorbance near 1700 cm-1. In the B-form, one class of protons is measured with an exchange half-time of 20 minutes. In the Z-form, two classes of protons are measured with very different exchange half-times, the exchange half-time of the slow protons being of the order of 850 minutes. By comparison of these results with those previously obtained for poly(dG-dC).poly(dG-dC), these very slow protons of these two Z-polynucleotides are identified as the cytosine amino protons. A quantitative description of the dynamic structure of the Z-form is presented.

Poly(dG-dC) . poly(dG-dC) was modified by chlorodiethylenetriamino platinum (II) chloride, cis-dichlorodiammine platinum (II) and trans-dichlorodiammine platinum (II), respectively. The conformation of these modified poly(dG-dC) . poly(dG-dC) was studied by circular dichroism. In 4 M Na+, the circular dichroism spectra of poly(dG-dC)dien-Pt (0 less than or equal to rb less than or equal to 0.2) are similar (rb is the amount of bound platinum per base). It is concluded that the conformation of these polymers belongs to the Z-family. Dien-Pt complexes stabilize the Z-form. The midpoint of the Z goes to B transition of poly(dG-dC)dien-Pt(0.12) is at 0.2 M NaCl. Moreover another B goes to Z transition is observed at lower salt concentration (midpoint at 6 mM NaCl). In 1 mM phosphate buffer, the stability of Z-poly(dG-dC)dien-Pt(0.12) is greatly affected by the presence of small amounts of EDTA. Poly(dG-dC) . poly(dG-dC) modified by cis-Pt and trans-Pt complexes do not adopt the Z-form even in high salt concentration.

The purpose of this work was to analyse at the molecular level the DNA recognized by the antibodies to Z-DNA in in situ experiments. Antibodies to Z-DNA interact strongly with R-band positive heterochromatic segments of fixed metaphase chromosomes of Cebus (Viegas-Pequignot et al., 1983). These segments are constituted of a satellite DNA the repeat unit of which is about 1520 base pairs long. The base sequence of the repeat unit has been determined. It contains a (AC)n rich region which, in vitro, adopts the Z conformation under topological constraints. Experiments with nuclei suggest that this sequence is not predominantly in the Z conformation in vivo. The polymorphic structure of the (AC)n rich region argues for an active recombination sequence.

Poly d(G-C) d(G-C) in B-form, on one hand, and poly d(G-br5C). poly d(G-br5C) and poly d(G-m5C) . poly d(G-m5C) in Z-form, on another hand, were treated with N-AcO-[3H]AAF and the kinetics of these reactions were followed by radioactivity. Covalent binding of carcinogen to the polymers was evaluated after separation of the reacted polymers from non-reacted carcinogen by thin-layer chromatography. We found that B-form polymer reacts twice faster than the Z-form polymers. Proportions of main adducts in the three polymers are almost the same. Results are discussed in relation to the calculated electrostatic potential minima and steric accessibility at the reactive site (1, 2).