1. Volume 4, Issue 6, Pages 1079-1085 (December 1999)
Inhibition of FEN-1 Processing by DNA Secondary Structure at Trinucleotide Repeats 
Craig Spiro, Richard Pelletier, Michael L Rolfsmeier, Michael J Dixon, Robert S Lahue, Goutam Gupta, Min S Park, Xian Chen, S.V.Santhana Mariappan, Cynthia T McMurray 
Molecular Cell 
Volume 4, Issue 6, Pages (December 1999)
DOI: /S (00)

2. Figure 1 Secondary Structure Increases the Rate of Expansion
(A) Flap inhibition model for expansion. Structure formation in flap prevents processing by the flap endonuclease, FEN-1.
(B) Selection scheme for expansion in yeast (Miret et al. 1998).
(C) Rates of expansion for wt and rad27Δ strains. Rates are expressed as the mean ± SD per generation. Each triplet is present in 25 copies; ranC,A,G refers to a randomized C,A,G sequence that is identical in base composition but contains no repeats. Sequences refer to the indicated trinucleotide in the lagging daughter strand orientation. Rates for the wild type except for GAC are from Miret et al
Molecular Cell 1999 4, DOI: ( /S (00) )

3. Figure 2 Stable Secondary Structures Form within Flap Templates
(A) Schematic structure of the three-stranded flap template; Fbr is the bridge oligonucleotide, Fadj is adjacent to the flap, and Flap contains the repeat and a sequence complementary to a portion of Fbr.
(B) The sequence of Fbr, Fadj, and CNG20 Flap templates used in NMR studies.
(C–F) The 500 MHz 1D NMR spectrum for imino protons in CTG20 (left) or CGG20 (right)-containing structures (1 mM in flap DNA, 10 mM phosphate [pH 6.9], 5°C). (C and E, top) Shown are 1D imino spectra for the CNG repeats contained within the hairpin alone (CAG20(C) or CGG20(E)) or within the flap template (FlapCAG20(C) or FlapCGG(C)). GC and AT indicate the imino proton in a Watson-Crick GC and AT pair, respectively; TT(C) and GG(E) represent the imino proton of the TT and GG mispaired bases, respectively. The 2D NOESY spectrum for imino protons in (FlapCTG20)(D) or (FlapCGG20)(F).
Molecular Cell 1999 4, DOI: ( /S (00) )

4. Figure 3 Secondary Structure Inhibits Flap Cleavage
(A) Schematic templates for three-stranded flaps in the presence (left) or the absence (right) of structure.
(B) Time dependence of FEN-1 cleavage of flaps comprising random sequence, no structure (Flap-1); CAG20, hairpin (Flap CAG20); and a repeat with no structure (Flap GTT). Added to each 20 μl reaction is 100 fmol of substrate and 1.2 ng of purified human FEN-1 incubated for the indicated time at 30°C.
(C) The effect of structure on FEN-1 inhibition of cleavage. Substrates and enzymes were incubated for 30 min at 30°C. Substrates comprised the indicated repeats within the flap.
(D) Effects of repeat length and sequence on FEN-1 cleavage.
(E) Representative gel separation of flap template cleavage products from a range of repeats, as indicated. Ramps indicate increasing FEN-1 in a 20 μl reaction (0, 0.3, 1.5, or 6.0 ng). Arrows indicate the position of cleavage products.
(F) Schematic diagram of flap templates comprising 1-, 2- and 3-strand oligonucleotides that do (left) or do not (right) form structure.
(G) Dissociation constants of FEN-1 binding to the flap structure. KD values for 1-, 2-, and 3-strand oligonucleotide templates (top, middle, and bottom rows, respectively) are indicated (for substrates in F). CCG, CTG, and CAG form structures; (randomized) rCAG and GTT form no structure. KD's corresponding to two concentrations (1.86 μM [top] and 0.74 μM [bottom]) of FEN-1 are shown.
Molecular Cell 1999 4, DOI: ( /S (00) )

5. Figure 4
Test of the Flap Inhibition Model for Expansion in Wild-Type and rad27Δ Strains
(A) A representative gel separation of PCR products for expanded CAG25 or CTG25 templates in a rad27Δ strain. S is the starting strain containing 25 repeats.
(B) The size and frequency of the expansion in wild-type and rad27Δ strains; black bars are wild type and gray bars are rad27Δ.
(C) Model for expansion by simple flap inhibition by secondary structure. Structure inhibits FEN-1 processing, and the increase occurs after flap ligation to the preceding Okazaki fragment. In this model, the size of the expansion is limited to twice the original allele size.
(D) A model for expansion by strand invasion mechanism. Flap structures facilitate expansion by recombination or single strand, template-directed synthesis. In this model, the size of the expansion can exceed twice the original allele size.
Molecular Cell 1999 4, DOI: ( /S (00) )