Journal: Nucleic Acids Research
Article Title: Mrc1 and Tof1 prevent fragility and instability at long CAG repeats by their fork stabilizing function
Figure Lengend Snippet: Analysis of replication through CAG-70 and CAG-130 repeats by two-dimensional (2D) agarose gel electrophoresis in WT, tof1 Δ and mrc1 Δ strains. ( A ) Schematic of the pYES2 constructs is shown with its mass and genetic map. The relative positions of its most relevant features are indicated inside: the 2 μm origin, the ColE1 unidirectional origin (ColE1 Ori), the ampicillin-resistance gene (Amp R ), URA3 , the GAL1 promoter (Gal1 prom) and 70 or 130 CAG repeats. Outside, the relative positions of sites recognized by the restriction endonucleases Nde I and BciV I are indicated. To the right, is shown the corresponding linear map of the pYES2 plasmid restriction fragment with the sizes and the diagrammatic interpretation if replication initiates bi-directionally at the 2 μm origin and proceeds unconstrained. ( B ) Representative 2D gels of replication through CAG-70 and CAG-130 repeats in WT, tof1 Δ, and mrc1 Δ strains. DNA was isolated, digested with Nde I and BciV I and analyzed by 2D gel. Red arrow points to the location of the CAG repeats. To the right of each 2D gel are shown the densitometric profiles corresponding to the Y-arc region where the (CAG)n repeats are located; peaks on densitograms correspond to bulges on the Y-arcs. A representative gel and its corresponding profile is shown; three experiments were analyzed for each strain. ( C ) Quantification of replication fork slowing in pYES2 CAG-130 in WT, tof1 Δ and mrc1 Δ strains. The ratio of radioactivity in the peak area to that corresponding area of a smooth replication arc reflects the extent of replication slowing. Three different experiments were performed for each strain. Percentage of replication fork slowing is 3.3%, 4.6% and 5.8% for WT, 13.2%, 17.7% and 20.4% for tof1 Δ, and 8.3%, 16.4% and 27.7% for mrc1 Δ. Error bars indicate standard error of the mean. The star indicates a significant difference between wild-type and mutants. P = 0.0483 ( tof1 Δ versus WT), P = 0.0378 ( mrc1 Δ versus WT).
Article Snippet: DNA was digested by Nde I, BciV I and Psi I (New England Biolabs) for 7 h at 37°C.
Techniques: Agarose Gel Electrophoresis, Construct, Plasmid Preparation, Isolation, Two-Dimensional Gel Electrophoresis, Radioactivity