ATRX dysfunction Induces replication defects in primary mouse cells

The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells

The structure and function of the chromatin remodelling domain of ATRX

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ATRX deficient cells show an increase in replication fork stalling and DNA double strand breaks.

<p>(A) Representative image of actual fibres from <i>Atrx^flox</i> mES cells. (B) Replication fork processivity in <i>Atrx^flox</i> and <i>Atrx^null</i> mES cells shown as a box whisker plot of the ratio in length of IdU and CldU labelled DNA for individual replicons (n = 259 for Flox and 359 for Null). Statistical significance was determined using a Mann Whitney test. (C) Representative images of five classes of replication intermediates identifed by DNA fibre analysis in this study. (D), (E) Relative frequency of replication intermediates in <i>Atrx^flox</i> and <i>Atrx^null</i> mES cells without (D) and with hydroxyurea treatment (E) during the IdU pulse. Over 1000 fibres totalled from three independent replicates were scored per experiment and error bars indicate ± SEM.</p

Loss of ATRX does not affect telomere maintenance in mouse ES cells.

<p>(A) Representative image of a telomere FISH performed on a metaphase spread. (B) Quantitation of telomere fluorescence intensity in Atrx<i>^flox</i> and Atrx<i>^null</i> mES cells from >7000 telomeres for each cell type. (C) Terminal restriction length analysis of two independent clones (1, IF12 and 2, IG11) after digestion with HinfI and RsaI. Digested DNA was probed with a radiolabelled telomeric repeat.</p

Loss of ATRX triggers a DNA damage response.

<p>(A) Representative images for immuno-FISH analysis showing colocalisation between 53BP1 and telomeres. Full data sets are available at <a href="http://sara.molbiol.ox.ac.uk/public/staylor/53BP1_Tel2" target="_blank">http://sara.molbiol.ox.ac.uk/public/staylor/53BP1_Tel2</a> and <a href="http://sara.molbiol.ox.ac.uk/public/staylor/53BP1_Tel3" target="_blank">http://sara.molbiol.ox.ac.uk/public/staylor/53BP1_Tel3</a>. (B),(C) 53BP1 foci and 53BP1 telomere colocalising foci were scored using the JACoP plugin for ImageJ from a total of 105 <i>Atrx^flox</i> and 115 <i>Atrx^null</i> cells. Statistical significance was determined using a Mann Whitney test. (D) Representative images of COMETs. (E) Quantitation of DNA DSBs by COMET assay represented by the proportion of DNA in the COMET “tail” (n = 50). Exposure to gamma-irradiation was used as a positive control. (F) Cellular sensitivity of <i>Atrx^flox</i> and <i>Atrx^null</i> mES cells to ionising radiation (IR), hydroxyurea (HU), Aphidicolin and Cisplatin as measured by clonogenic survival assay. Error bars indicate ± SEM from a minimum of three independent experiments.</p

ATRX interacts with the MRN complex in replicating cells.

<p>(A) Coomassie blue stained gel showing proteins immunoisolated from HeLa nuclear extract using anti-ATRX (H300) antibody. ‘No ab’ was performed using protein A beads only. HeLa nuclear extract pre-treated with λ phosphatase was used to assess phosphorylation dependence of the interaction. (B) Table showing identification of the three major proteins immunoisolated by mass spectrometry in the different IPs. Peptides were matched on the Mascot search engine for each of the proteins. (C) Immunoblots confirming presence of RAD50, ATRX, MRE11 and NBS1 in the proteins immunisolated by the ATRX antibody from HeLa nuclear extract. As a positive control a second reverse immunoprecipitation was performed using a RAD50 (H300) polyclonal antibody. ‘No IP’ was performed using protein A beads only. (D), (E) Representative images for immunofluorescence showing co-localisation between ATRX and MRE11 using C-terminal specific (D) and N-terminal specific (E) ATRX antibodies. (F) Representative images for immunofluorescence showing co-localisation between RAD50 and ATRX and 3 way co-localisation with PCNA (G). Full data set for the co-localisation of ATRX/RAD50 in HeLa cells is available at <a href="http://sara.molbiol.ox.ac.uk/public/staylor/ATRX_MRN_PCNA/" target="_blank">http://sara.molbiol.ox.ac.uk/public/staylor/ATRX_MRN_PCNA/</a>.</p