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

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

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

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

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

Doubling time of cells.

<p>Doubling time of cells.</p

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2′-deoxycytidine level in cultured cancerous cell lines

<div><p>Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2′-deoxycytidine, 5-(hydroxymethyl)-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxy-2′-deoxycytidine as well as 5-(hydroxymethyl)-2′-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt’s lymphoma lymphoblasts (Raji), EBV-negative Burkitt’s lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo). The expression levels of <i>TET1</i>, <i>TET2</i>, <i>TET3</i>, <i>SMUG1</i>, and <i>TDG</i> genes were also assayed by RT-qPCR. Our results show a global erasure of 5-hydroxymethyl-2′-deoxycytidine and 5-carboxy-2′-deoxycytidine in DNA of cultured cells compared with DNA from primary malignant tissue. Moreover, malignant cells in culture have a quite different DNA epigenetic profile than cultured normal cells, and different types of malignant cells display different and characteristic profiles of DNA epigenetic marks. Similar analyses of a broader spectrum of epigenetic modifications, not restricted to 5-methyl-2′-deoxycytidine, could lead to better understanding of the mechanism(s) responsible for emergence of different types of cancer cells.</p></div

A positive correlation between 5-(hydroxymethyl)-2′-deoxycytidine and mRNA expression level of <i>TET1</i> gene in malignant cell lines.

<p>A positive correlation between 5-(hydroxymethyl)-2′-deoxycytidine and mRNA expression level of <i>TET1</i> gene in malignant cell lines.</p

Levels of transcripts of the <i>TETs</i>, <i>SMUG1</i> and <i>TDG</i> genes in various cell lines.

<p>(A) <i>TET1</i>. (B) <i>TET2</i>. (C) <i>TET3</i>. (D) <i>SMUG1</i>. (E) <i>TDG</i>.</p

Levels of 5-mdC and intermediate products of active DNA demethylation in DNA from normal fibroblasts and various malignant cell lines.

<p>(A) Level of 5-mdC. (B) Level of 5-hmdC. (C) Level of 5-fdC. (D) Level of 5-cadC. (E) Level of 5-hmdU.</p