Journal: Journal of Cell Science

Article Title: Modified chromosome structure caused by phosphomimetic H2A modulates the DNA damage response by increasing chromatin mobility in yeast

doi: 10.1242/jcs.258500

Figure Lengend Snippet: Mimicking H2A S129 phosphorylation increases NHEJ and decreases translocation rates. (A) Schematic of the NHEJ assay principle. A replicative plasmid carrying HIS3 as an auxotrophic marker (pRS413) is linearized by EcoRI enzyme (cs, cut site) and used for yeast transformation. In strains with efficient NHEJ, plasmid extremities are joined (arrow) and His+ colonies are recovered on plates lacking histidine. (B) Rejoining efficiency was calculated for the indicated strains as the percentage recovery of His+ colonies after transformation of a linearized plasmid, relative to recovery following introduction of a non-linearized plasmid. A Δyku70 mutant (Ku70) is impaired in NHEJ and was used as a control for plasmid digestion efficiency. The bar graph shows the mean±s.e.m. of five experiments. **P≤0.01; ***P≤0.0001; n.s., not significant (non-parametric t-test). (C) Schematic of the strain used to measure translocation. Two DSBs generated by HO endonuclease (HO cs), an intronic sequence (dotted line) and truncated forms of URA3 are depicted on two distinct chromosomes (Chr. III and Chr. V). Reciprocal end-joining produces Ura+ cells. AI, artificial intron. (D) Percentage translocation efficiency for the indicated strains was calculated as the ratio of the number of Ura+ colonies recovered after persistent induction of DSBs (on −URA plates containing galactose) relative to the number recovered in non-translocated conditions (+URA plates containing galactose). The bar graph shows the mean±s.d. of five experiments. **P≤0.01; ***P≤0.0001; n.s., not significant (non-parametric t-test).

Article Snippet: Plasmid repair assay The plasmid pRS413 ( HIS3 ; Addgene) was linearized with EcoR I, which generates cohesive ends.

Techniques: Phospho-proteomics, Translocation Assay, NHEJ Assay, Plasmid Preparation, Marker, Transformation Assay, Mutagenesis, Control, Generated, Sequencing

Journal: Biochemical pharmacology

Article Title: Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells.

doi: 10.1016/j.bcp.2010.05.009

Figure Lengend Snippet: Fig. 1. Schematic overview of the conversion of TdR to TdR-related sugars and possible pathways which can stimulate angiogenesis. (A) TdR enters the cell, after which it is converted to thymine and deoxyribose-1-phosphate (dR-1-P). dR-1-P can be further converted to dR or dR-5-P. dR-5-P can enter the pentose phosphate pathway or glycolysis, stimulating the metabolism of the cells, possibly activating angiogenesis. dR is secreted by the cells, after which it can attract endothelial cells and/ or be taken up by the endothelial cells, stimulating angiogenesis. Both dR and dR-5-P can undergo Schiff base reactions, first by opening the ring structure of the sugar by anomerization, after which it can undergo a Schiff base reaction where an Amadori product is formed. Subsequently, reactive oxygen species (ROS) are formed, or advanced glycation end products (AGE). Moieties at which [50-3H]-TdR was labeled and the TdR-derived sugars are indicated with asterisk. TP: Thymidine phosphorylase; 1. thymidine kinase (TK); 2. DNA polymerase; 3. a phosphatase; 4. phospho-pentomutase; 6. deoxyribosephospatealdolase. (B) Structural formulae of the open ring structure of dR (2-deoxy-D-ribose), a Schiff base and an Amadori product. The open chain aldehyde glycates protein by a non-enzymatic condensation of the sugar to form a Schiff base, which rearranges to form an a- hydroxyketone Amadori product. dR-5-P can undergo the similar reaction. R = protein (see also ref. [13]).

Article Snippet: The thymidine phosphorylase antibody was purchased from R&D Systems Inc. (Minneapolis, MN, USA) and the thymidine kinase antibody was purchased from QED Bioscience (San Diego, CA, USA).

Techniques: Labeling, Derivative Assay

Journal: Biochemical pharmacology

Article Title: Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells.

doi: 10.1016/j.bcp.2010.05.009

Figure Lengend Snippet: Fig. 2. Western blot showing the expression levels of thymidine phosphorylase in Colo320, Colo320 TP1, RT112 and RT112/TP cells.

Article Snippet: The thymidine phosphorylase antibody was purchased from R&D Systems Inc. (Minneapolis, MN, USA) and the thymidine kinase antibody was purchased from QED Bioscience (San Diego, CA, USA).

Techniques: Western Blot, Expressing

Journal: Biochemical pharmacology

Article Title: Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells.

doi: 10.1016/j.bcp.2010.05.009

Figure Lengend Snippet: Fig. 3. The accumulation of sugars after conversion of TdR by thymidine phosphorylase. (A) Total levels of thymine and total levels of measured sugars. (B) Measurement of the levels of the various TdR-related sugars. (C) Measurement of the levels of dR that was secreted by the cells after degradation of TdR. All values represent means of three independent experiments SEM. Significant differences compared to the control levels are indicated *P < 0.05.

Article Snippet: The thymidine phosphorylase antibody was purchased from R&D Systems Inc. (Minneapolis, MN, USA) and the thymidine kinase antibody was purchased from QED Bioscience (San Diego, CA, USA).

Techniques: Control