Journal: Nucleic Acids Research
Article Title: Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast
Figure Lengend Snippet: Overproduction of Din7 enhances 5′−3′ dsDNA exonuclease activity in mitochondria. ( A ) Principle of the 5′−3′ and 3′−5′ exonuclease activity assay. The 5′- and 3′- 32 P-labels were preferentially removed by 5′−3′ and 3′−5′ exonuclease activity, and they are resistant to 3′−5′ and 5′−3′ exonucleases, respectively. ( B ) Schematic diagram of the construction of the Din7 deletion mutant (Din7Δ). Immunoblot analysis of overproduced C-terminally 6× His-tagged Din7 or Din7Δ in mitochondrial extracts (bottom). Mitochondria were isolated from cells bearing WT/pYES2/CT, WT/pYES2/CT-DIN7 and WT/pYES2/CT-din7Δ (overproducing Din7Δ with a C-terminal 6× His tag). The signals of overproduced Din7 and Din7Δ were detected using a monoclonal anti-6× His-tag antibody. As a control, the levels of porin (a constitutively expressed protein, used here as a control) were determined by immunoblot analysis using a monoclonal anti-porin antibody to adjust the protein concentrations of mitochondrial extracts from all three strains to the same level. ( C and E ) Analyses of 5′−3′ or 3′−5′ exonuclease activity in mitochondrial extracts. The 5′- or 3′- 32 P-labeled pUC119/HincII dsDNA fragments were incubated at 37°C for 15 min with increasing concentrations of mitochondrial extracts. After removing the proteins by treating the extracts with proteinase K, the DNA molecules were separated on a 1% agarose gel. ( D and F ) Quantitative representation of the 5′−3′ and 3′−5′ exonuclease activities in mitochondrial extracts, detected in (C) and (E). Signals relative to untreated DNA are plotted. ds, double-stranded; ss, single-stranded. Open circles, incubated with mitochondrial extracts from wild-type cells (cells without Din7 overproduction); closed circles, incubated with mitochondrial extracts from Din7-overproducing cells; closed triangles, incubated with mitochondrial extracts from Din7Δ-overproducing cells. Each bar represents the results of at least two independent experiments. ( G ) The generation of single-stranded DNA by Din7. HincII-linearized pUC119 DNA (104 µM) was treated at 37°C for 5 min with increasing amounts of mitochondrial extracts derived from cells of WT/pYES2/CT, WT/pYES2/CT-DIN7 or WT/pYES2/CT-din7Δ in the same buffer used for detection of λ exonuclease activity. Then, each reaction solution (10 µl) was separated into duplicate aliquots. Proteinase K was added to one aliquot to stop the reaction. The second aliquot from each set was treated at 37°C for 10 min with 2.25 U mung bean nuclease followed by addition of proteinase K to stop the reaction. Mung bean nuclease digests only the single-stranded DNA, leaving the double-stranded DNA intact. Samples were then electrophoresed on a 1% agarose gel and stained with ethidium bromide. Lane 1, 1-kb plus ladder used as a size marker; lane 2, HincII-linearized pUC119 DNA; lane 3, HincII-linearized pUC119 DNA treated in a reaction mixture without mitochondrial extracts; lanes 4–7, HincII-linearized pUC119 DNA treated with increasing amounts of mitochondrial extracts; lane 8, HincII-linearized pUC119 DNA treated only with mung bean nuclease; lanes 9–12, samples treated as in lanes 4–7, treated additionally with mung bean nuclease. The samples treated with mitochondrial extracts overexpressing DIN7 contain both single-stranded DNA fragments with discrete sizes and double-stranded DNA because discrete DNA signals smaller than those of the double-stranded DNA in the middle were removed by mung bean nuclease treatments.
Article Snippet: Assay for exonuclease activities in mitochondria The 5′-labeled pUC119/HincII dsDNA fragments (75 nM) and the 3′-labeled pUC119/BamHI dsDNA fragments (87 nM) were incubated at 37°C for 15 min with various amounts (0, 4, 20, 32 and 50 µg/ml in protein) of mitochondrial extracts in a 10-µl reaction solution containing 1× λ exonuclease buffer (New England BioLabs Inc., MA, USA): 67 mM glycine–KOH (pH 9.4), 2.5 mM MgCl2 and 50 µg/ml of bovine serum albumin.
Techniques: Activity Assay, Mutagenesis, Isolation, Labeling, Incubation, Agarose Gel Electrophoresis, Derivative Assay, Staining, Marker