Immunochemical detection of endogenous XRCC1 and PCNA at DNA repair sites

<p><b>Copyright information:</b></p><p>Taken from "XRCC1 and PCNA are loading platforms with distinct kinetic properties and different capacities to respond to multiple DNA lesions"</p><p>http://www.biomedcentral.com/1471-2199/8/81</p><p>BMC Molecular Biology 2007;8():81-81.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039748.</p><p></p> Widefield fluorescence images of Hela cells are shown. Cells were fixed at indicated time points after laser microirradiation. (A) Both, XRCC1 and PCNA, accumulate at laser-induced DNA damage sites. (B) Microirradiated Hela cells were extracted with 0,5% Triton-X100 and 500 mM NaCl prior to fixation. After in situ extraction no endogenous XRCC1 can be detected at microirradiated sites while PCNA accumulations can still be observed. Scale bars, 5 μm

Basic Principle of the Protein Molecular Force Assay.

<p>(A) Molecular Force Probes (MFPs) consist of two bonds in series, a protein complex to be studied and a DNA duplex acting as a reference. Both proteins are attached covalently at their N- or C-terminus, one to the glass slide and the other one to a strand of the DNA duplex. Cy5 and Cy3, coupled to one of the DNA strands each, form a FRET pair. Linkage to the upper surface, a PDMS stamp functionalized with Streptavidin, is facilitated <i>via</i> a Biotin on the DNA. In the macroscopic view, the PDMS stamp with 16 pillars as well as the glass slide with a matching 4×4 array of spots of MFPs is displayed. Every spot may be functionalized with a different set of MFPs, allowing for the measurement of 16 different protein pairs and/or the variation of the reference. (B) <i>Preparation:</i> After the stepwise assembly of the MFPs on the glass slide, fluorescence “Start” images of the Cy5 signal (with red excitation) as well as the FRET of the MFPs are recorded. Assembly of the assay is completed by lowering the stamp, which enables the Biotins of the MFPs to bind to the Streptavidins on the elastomer. <i>Force Assay</i>: Upon retraction of the stamp with constant speed, a force is gradually built up in the MFPs, acting equally on all molecular components in series. As a result, either the DNA reference duplex or the protein-protein interaction unbinds, resulting in the transfer of either Cy3 alone or Cy3 together with Cy5 to the surface of the stamp. <i>Readout</i>: Another set of fluorescence “Final” images of the glass surface provides the ratio of broken protein to reference bonds. The ratio of the Cy5 signals on the glass slide provides the surface density of remaining, intact protein complexes in comparison to the initial number of protein pairs. The residual FRET signal accounts for complexes that were not loaded under force and are still fully assembled. The ratio of the FRET signal thus allows for the correction of the analysis.</p

Utilization of Modified Reference DNA Duplexes to Adjust the Sensitivity Window in a Multiplexed Protein-MFA.

<p>(A) Three different reference types are compared: unmodified DNA (left), intrinsically stabilized DNA (center), where a part of the pyrimidine bases is replaced by corresponding propynyl bases, as well as extrinsically stabilized DNA (right), where the addition of a specific polyamide ligand <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115049#pone.0115049-Ho1" target="_blank">[23]</a> enhances the binding strength. (B) Representative sample measurements of Enhancer and Modified Enhancer binding to sfGFP for all types of references are displayed. The NF shows a clear dependence on the reference strength. The NF is higher for the Modified Enhancer than Enhancer in all cases. Additionally, the difference in NF between Modified Enhancer and Enhancer increases the closer the NFs are to 0.5, displaying the higher sensitivity in this range.</p

Analysis of Different GFP Variants for Enhancer Interaction Strength with Protein-MFA.

<p>(A) Schematic depiction of the MFP for the measurement of the interaction between GFP and Enhancer with the ribbon model structure of wtGFP (green) with Enhancer (magenta) (crystal structure from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115049#pone.0115049-Kirchhofer1" target="_blank">[30]</a>, PDB file 3K1K). One example measurement depicts the differences in binding strength of Enhancer tested against enhanced, wild type, and superfolder GFP with the same reference DNA (20 bp DNA stabilized with polyamide P1). While the binding to eGFP and wtGFP lie within the same range, binding of Enhancer to sfGFP is distinctively stronger. All data points are determined in one single measurement process, derived as the mean of several protein spots and displayed with standard deviation error bars. (B) Sample histograms of MFP spots of Enhancer measured against all three GFP variants illustrate the extensive number of parallelized single-molecule experiments. The Normalized Fluorescence (NF) is determined by dividing the raw fluorescence images before and after transfer pixel-by-pixel (according to Equation 1), and fitting of a Gaussian to the resulting histogram of all pixel counts.</p

Identification of mouse Tet3 transcript variants encoding a CXXC domain.

<p>(A) Drawing illustrating the generation of alternative transcripts from the <i>Tet3/Cxxc10-1</i> locus. The positions of primers used in B are reported. The lower part reports a schematic representation of alternative Tet3 transcripts. The positions of the probes used for northern blotting in C are reported. (B) Amplification of fragments from NSCs cDNA identifying Tet3 transcripts that include the Cxxc10-1 ORF. (C) Northern blot detection of alternative Tet3 transcripts in ESCs and NSCs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062755#pone.0062755.s001" target="_blank">Fig. S1</a> for full and additional blots).</p

Genomic arrangement of mouse <i>Tet</i> genes and adjacent <i>Cxxc</i> loci (A) and homology of CXXC domains from mouse Cxxc4, Cxxc5 and Tet homologues in various animal species (B).

<p>(A) Schematic representation of mouse <i>Tet1</i>, <i>Tet2/Cxxc4</i> and <i>Tet3/Cxxc10</i> loci. Exons are depicted as blue rectangles. Annotated transcriptional start sites and transcription orientation are indicated with half arrows. (B) Alignment of CXXC domains from mouse Cxxc4, Cxxc5 and Tet homologues in various animal species (Mm, <i>Mus Musculus</i>; Hg, <i>Heterocephalus glaber</i>; Xt, <i>Xenopus tropicalis</i>; Hm, <i>Hydra mangipallata</i>). The alignment was generated with Unipro UGENE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062755#pone.0062755-Okonechnikov1" target="_blank">[64]</a>. Numbers on the right side indicate the position of the last amino acid in the corresponding protein. The KTXXXI motif, previously identified as determinant for the interaction of Cxxc4 with Dvl <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062755#pone.0062755-London1" target="_blank">[54]</a>, is boxed (see Discussion). The scale at the bottom indicates the upper limit of percent identity represented by each color. GenBank accession numbers: MmCxxc10, JX946278; XtTet3, NP_001090656.1; HgTet3, EHB01729.1; MmTet1, NP_081660.1; MmCxxc4, NP_001004367; MmCxxc5, NP_598448; HmTet, XP_002161163.1.</p

Tet3 and Cxxc4 interact <i>in vivo</i>.

<p>The interaction was detected by the F3H assay in BHK cells harboring a <i>lac</i> operator array (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062755#pone.0062755.s002" target="_blank">Fig. S2</a> for explanations). (A) An N-terminal fusion of Tet 3 with Cherry (Ch) was used as prey and GFP-Cxxc4 (upper row) or GFP (as control; lower row) as baits. Localization patterns are representative of 8 (upper row) and 9 (lower row) out of 10 imaged cells. (B) Ch-Cxxc4 was used as prey and GFP-Tet3 (upper row) or GFP (as control; lower row) as baits. Localization patterns are representative of 4 out of 5 (upper row) and 6 out of 7 (lower row) imaged cells. Arrowheads indicate the position of the <i>lac</i> operator array as identified by bait signals (GFP channel). Scale bars: 5 µm.</p

Levels of Tet3, Cxxc4 and Cxx5 transcripts in mouse adult tissues, NSCs and ESCs.

<p>Transcript levels were determined by qPCR analysis of total cDNA. (A) Amplfied fragments identify the Tet3 mRNA refseq NM_183138 (Tet3), the alternative Tet3 transcript containing the Cxxc10-1 ORF and exon 2 of NM_183138 (Tet3^CXXCL) and all transcripts including the Cxxc10-1 ORF. (B) Cxxc4 and Cxx5 transcript levels. Data relative to kidney, liver, cerebellum and cortex samples are from three biological replicates (two 6 week old 129Sv mice and a 30 week old C57BL/6 mouse). Data relative to spleen, heart, lung and hippocampus are from two biological replicates (a 6 week old 129/Sv mouse and a 30 week old C57BL/6 mouse). Data relative to NSCs and ESCs are from three independent cultures each. Shown are mean values and standard errors of the mean (SEM).</p

<i>In vitro</i> DNA binding properties of Cxxc4 and 5, isolated CXXC domains and full length constructs of Tet1 and Tet3^CXXC.

<p>All proteins were expressed as GFP fusion constructs in HEK293T cells and affinity purified using a GFP-trap. Fluorescently labeled DNA substrates with the same sequence and a single CpG site either unmethylated, symmetrically methylated or symmetrically hydroxymethylated were incubated in direct competition. Shown are mean values of bound substrate/protein ratios and SEM from n independent replicate experiments: Tet1, n = 10; Tet3, CXXC^Tet3, n = 6; Tet3^CXXCL, n = 7; CXXC^Tet1, Cxxc4 and GFP, n = 3; Cxxc5, n = 2.</p

Automated monitoring of HIV morphogenesis.

<p>(<b>A</b>) HeLa-Kyoto cells, stably expressing CA_NTDcb1eGFP were transfected with pcHIV, encoding untagged Gag and monitored by time lapse epifluorescence microscopy for 18 h at 10 min time intervals. Scale bar, 50 µm. (<b>B</b>) Automated pattern recognition for High Content Analysis. eGFP fluorescence was used to determine borders of individual cells by intensity threshold segmentation, indicated by green lines. Particles within cells were segmented and defined by size and intensity threshold setting, indicated by cyan coloured lines. Scale bars, 50 µm. The average number of granules per cell was determined and plotted over time. Error bars represent the standard error of the mean (n∼400 cells).</p