Formation and repair of DNA double-strand breaks caused by ionizing radiation in the Epstein-Barr virus minichromosome

L’ADN dans nos cellules est exposé continuellement à des agents génotoxiques. Parmi ceux-ci on retrouve les rayons ultraviolets, les agents mutagènes chimiques d’origine naturelle ou synthétique, les agents radiomimétiques, et les dérivés réactifs de l’oxygène produits par les radiations ionisantes ou par des processus tels que les cycles métaboliques redox. Parmi les dommages infligés par ces agents, les plus dangereux sont les cassures simples- et double-brin de l’ADN qui brisent son intégrité et doivent être réparées immédiatement et efficacement afin de préserver la stabilité et le fonctionnement du génome. Dans la cellule, ces cassures sont formées et réparées au niveau de la chromatine, où l'environnement moléculaire et les évènements impliqués sont plus complexes et les systèmes expérimentaux appropriés pour leur exploration sont peu développés. L’objectif de ma recherche visait ainsi l’exploration de ces processus et le développement de nouveaux modèles qui nous permettraient d’étudier plus précisément la nature de la formation et de la réparation des cassures simple- et double-brin de l’ADN in vivo. J’ai utilisé comme modèle un minichromosome (l’episome du virus Epstein-Barr) d’environ 172 kb, qui possède toutes les caractéristiques de la chromatine génomique. Nous avons observé que la radiation gamma induit un changement conformationnel de l’ADN du minichromosome par la production d’une seule cassure double-brin (CDB) localisée de façon aléatoire. Une fois linéarisé, le minichromosome devient résistant à des clivages supplémentaires et par la radiation ionisante et par d’autres réactifs qui induisent des cassures, indiquant l’existence d’un nouveau mécanisme qui dépend de la structure chromatinienne et par lequel une première CSB dans le minichromosome confère une résistance à la formation d’autres cassures. De plus, la reformation des molécules d’ADN du minichromosome surenroulées après l’irradiation indique que toutes les cassures simple-brin (CSB) et CDB sont réparées et les deux brins fermés de façon covalente. Nos découvertes indiquent que la réparation par ligature d'extrémités d'ADN non homologues est le principal mécanisme responsable de la réparation des CDB, alors que la réparation des CSB est indépendante de la polymérase poly-ADP ribose-1 (PARP-1). La modélisation mathématique de la cinétique de réparation et le calcul des vitesses de réparation a révélé que la réparation des CSB est indépendante de la réparation des CDB, et représente l’étape limitante dans la réparation complète des minichromosomes. Globalement, nous proposons que puisque ce minichromosome est comparable en longueur et en topologie aux boucles d’ADN sous contrainte de la chromatine génomique in vivo, ces observations pourraient fournir une vision plus détaillée de la cassure et de la réparation de la chromatine génomique.DNA in our cells is exposed continually to DNA-damaging agents. These include ultraviolet light, natural and man-made mutagenic chemicals, and reactive oxygen species generated by ionizing radiation or processes such as redox cycling by heavy metal ions and radio-mimetic drugs. Of the various forms of damage that are inflicted by these mutagens, the most dangerous are the single- and double-strand breaks (SSBs and DSBs) which disrupt the integrity of DNA and have to be repaired immediately and efficiently in order to preserve the stability and functioning of the genome. In the cell, induction and repair of strand breaks takes place in the context of chromatin where the molecular environment and the events involved are more complex and suitable experimental systems to explore them are much less developed. A major focus of my research was therefore aimed towards exploring these processes and developing new models which will allow us to look more precisely into the nature of induction and repair of SSBs and DSBs in DNA in vivo. We used as a model the naturally-occurring, 172 kb long Epstein-Barr virus (EBV) minichromosome which posses all the characteristics of genomic chromatin and is maintained naturally in Raji cells. Gamma-irradiation of cells induces one, randomly-located DSB and several SSBs in the minichromosome DNA, producing the linear form. The minichromosome is then resistant to further cleavage either by ionizing radiation or by other break-inducing reagents, suggesting the existence of a novel mechanism in which a first SSBs or DSBs in the minichromosome DNA results in a conformational change of its chromatin which confers insensitivity to the induction of further breaks. Supercoiled molecules of minichromosome DNA were reformed when cells were incubated after irradiation, implying that all SSBs and DSBs were repaired and both strands were covalently closed. Using specific inhibitors or siRNA depletion of repair enzymes, we found that Non Homologous End Joining was the predominant pathway responsible for DSB repair, whereas repair of SSBs was PARP-1 independent. We could also show clearly that topoisomerases I and II are not required for repair. Mathematical modeling of the kinetics of repair and calculation of rate constants revealed that repair of SSBs was independent of repair of DSBs and was the rate-limiting step in complete repair of minichromosomes. Overall, we propose that since this minichromosome is analogous in length and topology to the constrained loops which genomic chromatin is believed to form in vivo, these observations could provide more detailed insights into DNA breakage and repair in genomic chromatin

Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ~170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20-30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair

Arrest of double strand break repair by inhibitors of DNA-PKcs phosphorylation.

<p>(A) Phosphorylation of DNA-PKcs on threonine-2609 (green) in cells irradiated and incubated without or with wortmannin (100 µM) or (C) without or with NU7441 (10 µM) assayed by immunofluorescence; DNA was stained by DRAQ (red). Below, quantitation of the signal from DNA-PKcs2609Thr-P (green pixel intensity/nuclear area). (B) Repair in cells incubated with wortmannin (100 µM) or (C) NU7441 (10 µM). (D) Quantitation of linear and supercoiled DNA during repair. Error bars show SEM from three independent experiments, or two independent experiments for NU7441.</p

Repair of single strand breaks in linear minichromosome DNA.

<p>(A) DNA synthesis (incorporation of [¹⁴C]thymidine) in irradiated and control cells in the conditions used for repair; error bars show SEM from three independent experiments. (B) Fragmentation by nuclease S1 of linear minichromosome DNA isolated immediately after irradiation (50 Gy) or after repair for 2 h. Linear DNA was isolated from a gel of total cell DNA and incubated without or with nuclease S1 for 15 h and the fragments produced were separated by PFGE. For these experiments sufficient linear DNA could be conserved for 2 h only if repair of double strand breaks was arrested; this was achieved by including the DNA-PK inhibitor NU7441 during repair as described in the Section "Pathways for repair of double strand breaks". (C) Scans of the hybridisation signal from lanes in (B) (nuclease S1 100 u/ml); the position of full-length linear molecules is indicated by the vertical dashed line.</p

Strand breaks in minichromosome DNA in irradiated cells.

<p>(A) Supercoiled minichromosome DNA and forms which result from strand breaks. (B) Minichromosome DNA separated by PFGE after incubating deproteinised cells with: lane C, no addition; lane PacI, PacI (100 u/ml, 3 h) which cuts minichromosome DNA at a single site; lane NbB, endonuclease Nb.BbvCI (100 u/ml, 1 h) which forms circular molecules containing single strand breaks. Lane 50 Gy, cells irradiated (50 Gy) before deproteinisation; lane λ, oligomers of λ DNA. The gel was hybridised with a probe of EBV DNA; for the gel images in this and following Figures the top includes the sample well and panels were assembled from lanes of the same gel. (C) Representative DNA molecules believed to be relaxed circular minichromosome DNA containing single-strand breaks, extracted from the region close to the origin of a gel of DNA from cells incubated with endonuclease Nb.BbvCI (panel B, lane NbB), stained with YOYO-1, and combed (see text). (D) Quantitation of linear minichromosome DNA in irradiated cells compared with that after cleavage at its single PacI site (100 u/ml, 3 h) in deproteinised cells; error bars show SEM from three independent experiments. (E) Representative linear minichromosome DNA from irradiated cells spread by molecular combing and hybridised with the two probes shown on the upper map; TR are the terminal repeat sequences by which the minichromosome is circularised. The probes were labeled with biotin and detected with anti-biotin antibodies (green), and DNA was labelled with BrdU and detected with anti-BrdU antibodies (red). The extremities of the molecules show the site of the double strand break; the probe positions were aligned approximately considering the slightly variable stretching of DNA during combing. (F) Linear minichromosome DNA from irradiated cells extracted from a gel, incubated without or with nuclease S1 (100 u/ml, 15 h), and subjected to PFGE.</p

Effect of inhibiting HR-mediated repair of double strand breaks.

<p>(A) Phosphorylation of ATM on Ser1981 (green) in cells irradiated and incubated without or with caffeine (10 mM) or KU55933 (20 µM), assayed by immunofluorescence; DNA was stained by DRAQ (red). Below, quantitation of the signal from ATM1981S-P (green pixel intensity/nuclear area). (B) Repair of minichromosome DNA in cells incubated without or with caffeine (10 mM) or KU55933 (20 µM), inhibitors of ATM kinase, or (C) with mirin (100 µM) which prevents activation of ATM without affecting its kinase activity. (D) Repair in cells transfected with siRNA to silence expression of Rad51 or with a control siRNA; cells were irradiated 48 h later and incubated for repair. Rad51 protein was detected in cell lysates by Western blot, with actin as a sample loading control. All error bars show SEM from three independent experiments.</p

Temporal evolution of the levels of different forms of minichromosome DNA during repair calculated by modeling.

<p>(A) The model considered transfers of molecules between four compartments containing supercoiled molecules (<i>S</i>), linear molecules formed by a double strand break (<i>L</i>), linear molecules also containing single strand breaks (*) (<i>LSSB</i>), and circular molecules containing single strand breaks (<i>CSSB</i>). <i>k_s, k_sd, k_d,</i> and <i>k_ds</i> are the rate constants, and <i>k_d,</i> and <i>k_ds</i> were set at zero when repair of double strand breaks was arrested by the inhibitor NU7441. (B, C) Calculated levels of the different forms of minichromosome DNA (curves) together with the experimental data points with SEM from three independent experiments, (B) during normal repair or (C) when repair of double strand breaks is arrested. (D) Calculated levels of the different forms of DNA extrapolated for a period of 20 h in normal conditions (full lines) or when the repair of double strand breaks is arrested (dashed lines).</p