1. Volume 3, Issue 4, Pages 465-475 (April 1999)
Sticky DNA 
Naoaki Sakamoto, Paul D. Chastain, Pawel Parniewski, Keiichi Ohshima, Massimo Pandolfo, Jack D. Griffith, Robert D. Wells 
Molecular Cell 
Volume 3, Issue 4, Pages (April 1999)
DOI: /S (00)

2. Figure 1
Agarose Gel Electrophoresis of Supercoiled, Insert-Excised, and Linearized Plasmids
(A) Supercoiled plasmids isolated from E. coli cells. A series of (GAA·TTC)n-containing plasmids (D) were analyzed in a 0.7% agarose gel.
(B) EcoRI-EcoRV-digested plasmids. The plasmids shown in (A) were digested with EcoRI and EcoRV and analyzed in a 1.5% agarose gel.
(C) SacI-linearized plasmids. The plasmids shown in (A) were digested with SacI and analyzed in a 0.7% agarose gel. The numbers of repeats are shown below the figure. 1 kb, 1 kb DNA ladder (GIBCO–BRL). The sizes (kbp) of the 1 kb DNA ladder are shown on the right of the gels.
(D) List of plasmids used in this study. The lengths of the repeat inserts from 9 to 59 were determined precisely by sequencing. For the inserts with lengths ≥75, restriction mapping was employed, and the lengths are estimated to be ±5. The length of the hexamer repeat is 65 ± 2 from DNA sequencing.
(E) Quantitation of the amount of retarded band as a function of GAA·TTC repeat length. The amount of the retarded bands in (C) was quantitated by densitometric analysis of the photographic negative of the gel. The total amount of DNA in each lane was defined as 100%. Solid circles, (GAA·TTC)n of various lengths (C and D); open square, (GAAGGA·TCCTTC)65. (GAAGGA·TCCTTC)65 was plotted as a 130 repeating triplet.
Molecular Cell 1999 3, DOI: ( /S (00) )

3. Figure 2
The Effect of the Location of (GAA·TTC)59 or (GAA·TTC)150 in Linearized Plasmids on the Extent of Retardation of Bands
pRW3821 or pRW3822 was linearized with one of five different restriction enzymes.
(A) Schematic figure of linearized pRW3821 or pRW3822 by one of five different restriction enzymes (right side). Open box, human flanking sequences; closed box, (GAA·TTC)59 or (GAA·TTC)150 repeat sequences.
(B) Linearization of pRW3821 or pRW3822 using one of the five different restriction enzymes. Supercoiled pRW3821 or pRW3822 was digested with the designated enzymes and analyzed in a 0.7% agarose gel. The enzymes used are shown on the top of the figure. 1 kb, 1 kb DNA ladder; sc, supercoiled plasmids. The positions of the expected linearized molecules and the retarded bands are shown on the right of the figure, and the sizes (kbp) of the 1 kb DNA ladder are shown on the left.
Molecular Cell 1999 3, DOI: ( /S (00) )

4. Figure 3 Properties of the Retarded Band
(A) Effect of negative supercoil density on the formation of the “42 kbp” retarded band. pRW3822 topoisomer families were prepared as described in the Experimental Procedures, and the supercoiled molecules were subjected to 0.7% agarose gel electrophoretic analyses. The sizes of the 1 kb DNA ladder are shown on the right.
(B) XmnI-digested pRW3822 topoisomers. The pRW3822 topoisomers shown in (A) were digested with XmnI and analyzed in a 0.7% agarose gel. 1kb, 1kb DNA ladder; sc, supercoiled pRW3822 isolated from E. coli. The numbers below the figure represent the concentrations of EtBr (μg/ml) used in the preparation of topoisomers. RB, retarded band; 7.1, 7.1 kbp linear pRW3822.
(C) Quantitation of the effect of superhelical density. The amount of the “42 kbp” retarded bands in (B) was quantitated by densitometric analysis. It was not possible to accurately calculate the superhelical density at 5.0 μg EtBr/ml.
(D) Effect of heat and/or EDTA on the amount of the “33 kbp” retarded band of EcoNI-digested pRW3822. EcoNI-digested pRW3822 was purified and dissolved in TE buffer (10 mM Tris–HCl [pH 8.0] and 1 mM EDTA) or in 10 mM Tris–HCl (pH 8.0). Plasmid solutions were incubated at different temperatures for 10 min and analyzed in a 0.7% agarose gel. The temperatures (°C) are shown on top of the picture. 1 kb, 1 kb DNA ladder; sc, supercoiled pRW3822. The sizes (kbp) of the 1 kbp ladder are shown on the left of the picture, and the positions of expected linear molecules (7.1 kbp) and the “33 kbp” retarded band are designated on the right.
(E) Quantitation of the effect of temperature and EDTA. The amount of the retarded bands in (D) was quantitated by densitometric analysis of the gel picture. The amount of the retarded band at each temperature was compared to 5°C, which was defined as 100%. Solid circles, in the presence of EDTA; open circles, in the absence of EDTA.
(F) Mapping of P1 nuclease-sensitive sites in pRW3822. Supercoiled pRW3822 (−σ = 0.055) was treated with P1 nuclease, and the cleavage sites in each of the strands were mapped as described in the Experimental Procedures. The relative intensity of the signals was quantitated by densitometric analysis of the autoradiograph. The location of the signals was determined by comparison with the migration of a 1 kb DNA ladder in the same gel. The nonalignment of the TRS sizes on the two strands was due to the requirement for opposite orientations of the complementary strands.
Molecular Cell 1999 3, DOI: ( /S (00) )

5. Figure 4
EM Visualization of the Isolated 7.1 kbp Band and the “42 kbp” Retarded Band
(A) Predominant structures found in the isolated 7.1 kbp band. pRW3822 was digested with XmnI, and the 7.1 kbp band and the “42 kbp” retarded band were separated by 0.7% agarose gel electrophoresis. DNA was isolated from each of the bands (Experimental Procedures) and analyzed by electron microscopy. The bar is 1 kbp in length.
(B) Predominant structures found in the “42 kbp” isolated retarded band.
(C) Quantitation of structures observed by electron microscopy in the isolated retarded band and the isolated linear band for plasmids containing (GAA·TTC)150 or (GAA·TTC)270.
(D) Placement of the DNA:DNA interaction within pRW3822. pRW3822 was linearized with XmnI and analyzed by measuring the distance from each DNA end to the beginning of the DNA:DNA interaction along with the DNA:DNA interaction itself. The gray bar shows the position of the DNA:DNA interaction. The black bar shows the position of the triplet repeat insert. Lengths were plotted relative to the average length of linear pRW3822 molecules. Inset, distribution of the lengths of DNA:DNA interactions.
Molecular Cell 1999 3, DOI: ( /S (00) )

6. Figure 5 EM Visualization of Associated Linear pRW3822 Molecules
pRW3822 was digested with one of the five designated enzymes (Figure 2) and analyzed by electron microscopy. The left side shows the schematic structures of the associated molecules from each digestion. Open boxes represent the (GAA·TTC)150 repeat. The percentages of the DNAs scored (150–321 for each of the five linearization reactions) in the dimeric-associated forms are listed in brackets. The right side shows typical electron micrographs of each digestion. The bar is 1 kbp in length.
Molecular Cell 1999 3, DOI: ( /S (00) )

7. Figure 6
EM Visualization of Two Molecules of Nicked Circular pRW3822 Associating with Each Other
The circular pRW3822 isolated from E. coli was analyzed by electron microscopy. The bar designates 1 kbp in length.
Molecular Cell 1999 3, DOI: ( /S (00) )

8. Figure 7
A Model of the Association of Two Triplexes Formed by Long GAA·TTC Tracts in Two Plasmids
(A) Schematic picture of the strand exchange model. The two triplexes are represented as thin and thick lines. The short vertical lines between the bases represent Watson-Crick pairs, and the stars represent the reversed Hoogsteen base pairs.
(B) Three-dimensional picture of the strand exchange model. Different colors represent different strands. In the left molecule, blue shows the purine strand, while yellow shows the pyrimidine strand. In the right molecule, green shows the purine strand and red shows the pyrimidine strand.
Molecular Cell 1999 3, DOI: ( /S (00) )