1. Strand Invasion Structures in the Inverted Repeat of Candida albicans Mitochondrial DNA Reveal a Role for Homologous Recombination in Replication 
Joachim M. Gerhold, Anu Aun, Tiina Sedman, Priit Jõers, Juhan Sedman 
Molecular Cell 
Volume 39, Issue 6, Pages (September 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1 Organization, structure and topology of C. albicans mtDNA
(A) The mtDNA of C. albicans has a unit size of 40.4 kb, is organized into a small and large coding region (SCR and LCR), and contains a 2 × 7 kb inverted repeat (IRa and IRb). Probes used in this study are indicated by black bricks and names, filled dark gray arrows indicate genes, and dashed light gray arrows indicate the IR.
(B) Restriction fragment analysis of mtDNA from C. albicans with single or rare cutting enzymes. Asterisks mark expected bands. Arrows mark bands corresponding to breaks in IR sequences. Two additional prominent bands show that SCR is present in two orientations as depicted in (D, black arrows in I and II). The probe used was cox3.
(C) PFGE of mtDNA probed for cox2 and exposed to a phosphorimager screen for 12 hr. Agarose-embedded whole cells, extracted mitochondria, and purified mtDNA were analyzed untreated or enzyme treated as indicated: lane 1, embedded cells; lane 2, embedded cells cleaved NcoI; lane 3, embedded mitochondria; lane 4, embedded mitochondria cleaved NcoI; lane 5, purified mtDNA; lane 6, purified mtDNA cleaved NcoI; lane 7, purified mtDNA treated with TopoI; and lane 8, purified mtDNA treated with T7EndoI. The compression zone (cz) is indicated by an arrow. Even after a prolonged 3 day exposure, no signal could be attributed to circular DNA.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Replication and Recombination Intermediates of C. albicans mtDNA
(A–F) 2D-AGE of digested mtDNA purified from C. albicans mitochondria (A–E; restriction enzymes and probes are indicated), interpreted (F) (C, cloud arc; X, X arc, Y, Y arc). Y structures, X structures, and diffuse arcs of complex and branched molecules (cloud arc “C”), can be observed. Strong signals are detected on the arc of linear dsDNA below 1N.
(G) Quantifications of 2D-AGE radiographs from (A)–(E). Data are shown as average hybridization signal distribution into specified areas of the analyzed radiographs (± standard error [SE]).
(H) Quantifications were performed by division of radiographs into four areas; reaching from the well to the X arc defined as “cloud,” “Y and X” covering X arcs and Y arcs, 1N spot, and signal on the dsDNA-arc below 1N.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Specific Y-Shaped Molecules Indicate Recombination between IRa and IRb of the C. albicans mtDNA
(A–F) 2D-AGE of mtDNA digested with DraI (A and E), DraI-NcoI (B), BamHI-NcoI (C), and BamHI-EcoRI (D) spanning fragments containing sequences of SCR and IRa/IRb or only IRa/IRb (E). Black arrows in (A)–(E) indicate the 2N spot of regular Y arcs. Panels (A)–(D) reveal extra small or extra large additional Y arc patterns (interpreted in F) (C, cloud arc; X, X arc; Y, Y arc; YES, extra small Y arc; YEL, extra large Y arc).
(E) A DraI fragment containing only IR sequence does not reveal YES/YEL arcs or bubble structures.
(G and I) Depending on the unique positions of restriction sites within SCR, the arcs form as a result of HR between IRa and IRb; X indicates homologous pairing and strand invasion, and a filled black dot marks the position of a probe used to detect the specific Y structures (G). A restriction map is in (I).
(H) YES and YEL structures are predicted to form via 3′ ssDNA overhang invasions into homologous sequences.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
RNase H and S1 Nuclease Treatments Indicate RNA:DNA Duplexes and ssDNA-Containing Structures
C. albicans mtDNA was incubated with or without RNaseH, digested with EcoRV and treated with or without S1 nuclease prior to 2D-AGE analysis (A–D, interpretation in E) (C, cloud arc; X, X arc; Y, Y arc; Q, quick-moving arc). Major parts of the C arc and the ssDNA arc are removed by S1 nuclease showing exposed ssDNA (B and D). RNaseH removed the “flag” structure and lead to formation of a distinctive S1-sensitive spot below the dsDNA arc close to the 1N spot (C). See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Fork Direction 2D-AGE of C. albicans mtDNA
(A) Opposing replication forks may form by simultaneous strand invasions at one locus (I) or at different loci (II).
(B and C) mtDNA digested with BglII/EcoRI (B) and DraI (C) was separated on a 1D gel, digested with EcoRV (B) or SpeI (C) in gelo, and then separated on a 2D gel (interpreted in D). Y arc patterns of both possible fork directions are observed on both radiographs showing that replication forks pass the fragments in opposing directions.
(D) Interpretation of fork direction 2D gels; dashed lines show the original 2D-AGE pattern, and heavy lines depict the obtainable pattern upon in gelo digest. Formation of the two possible patterns is shown below “Yb” and “Ya,” and the position of the probe is indicated by a black filled box above the Y structures. The vertical dotted line crossing the Y structures mimics the position of in gelo cleavage.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Relative mtDNA Copy Numbers of a C. albicans Mutant Strain Defective in mtDNA Maintenance in Comparison to WT mtDNA
(A) Total cellular DNA preparations of C. albicans WT strain CAI4 and hmi1-deletion strain PJ387 (Jõers et al., 2007) were dot blotted and hybridized to different probes (see also Figure 1A). The bar diagram is aligned with the map of the C. albicans mtDNA and the positions of probes. Names of probes are indicated above each bar. Bars show average relative mtDNA copy numbers in strain PJ387 to WT CAI4 (±SE). The horizontal line indicates wild-type level (100%). mtDNA signals were normalized against nuclear DNA.
(B) Average relative mtDNA copy numbers (±SE) at the ends of the inverted repeat sequences (nt 5540–6776 IRa and nt 39210–40420 IRb) as detected by probes R1–R9 (Table S1).
Molecular Cell  , DOI: ( /j.molcel )
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