1. Volume 1, Issue 4, Pages 583-593 (March 1998)
GAA Instability in Friedreich's Ataxia Shares a Common, DNA-Directed and Intraallelic Mechanism with Other Trinucleotide Diseases 
A.Marquis Gacy, Geoffrey M Goellner, Craig Spiro, Xian Chen, Goutam Gupta, E.Morton Bradbury, Roy B Dyer, Marci J Mikesell, Janet Z Yao, Aaron J Johnson, Andrea Richter, Serge B Melançon, Cynthia T McMurray 
Molecular Cell 
Volume 1, Issue 4, Pages (March 1998)
DOI: /S (00)

2. Figure 1 Expansion of GAA in Friedreich's Ataxia Is DNA-Directed
(A) Pedigree of a representative Friedreich's Ataxia family (AF20). The size of the frataxin GAA repeat on both alleles is listed. Capital letters indicate haplotypes for individual alleles as determined by sizing nine nearby nonrecombinant loci. In the two homozygous individuals, the size of the GAA repeat cannot be assigned to the haplotype because both alleles are unstable.
(B) Instability of repetitive loci in Friedreich's Ataxia families. Repeats were analyzed for changes in size during parent to offspring transmission: (a) Each family pedigree is listed with the total number of complete generations (#gen) and (b) the number of father-mother-offspring sets (FMO). (c) Repeating dinucleotide and trinucleotide repeats in 10 loci were analyzed for changes in repeat size during parent to offspring transmission. Percent instability is the number of unstable alleles divided by the total number of alleles, multiplied by 100. The other loci are as follows: DY, dynorphin; HD, huntington; SI, spino-cerebellar ataxia type I; TB, TATA binding protein; WG, WG0C5RPl; U0, U06699; SA, SADNA2; AC, ACTC; S2, D6S264; S3, D6S305. The repeat sequence at each locus is indicated. (d) The short (<30 GAA repeats) frataxin allele (FA[S]) is separated from the long frataxin allele (FA[L]) for purposes of instability analysis. (e) Analysis for CC and HD is summarized from Goellner et al. 1997; in a single HD patient modest instability was found at two loci (asterisk).
(C) Distribution of GAA repeat sizes in both the normal and expanded frataxin allele from the FRDA families studied.
(D) Distribution of the change in long (FA[L]) GAA repeat sizes. (E) Gender separation of part D: changes in allele size from mother to offspring (filled boxes) and changes in allele size from father to offspring (open boxes).
Molecular Cell 1998 1, DOI: ( /S (00) )

3. Figure 2
A Parallel YRY Triplex Forms in the Frataxin GAA/CTT Repeat Region
(A) Oligonucleotides used to study triple helix formation. Each sequence is a single contiguous oligonucleotide except (pY)·RY, which comprises two sequences, RY and (CTT)7.
(B) Thermal melting transitions provide evidence for triple helix formation of GAA/CTT repeats. Melting curves (at 260nm) (in 0.1M NaCl PIPES) correspond to the immediately adjacent sequences (in [A]). The RY melting transition (77°C) is indicated by the open arrow. The melting transition of the YRY third strand is indicated by either I (intrastrand) or II (interstrand).
(C) Molecular weight determination of oligonucleotides. Molecular weights (molecules from [A]) were calculated from the base composition (Calc MW). For (pY)·RY, the molecular weight of each component is indicated. Observed molecular weights were from sedimentation equilibrium.
(D) Derivative plots show distinct third strand and duplex melting transitions. The first derivative of the absorbance melting curves from (B) for pY·RY (left) and (pY)·RY (right) are shown; melting transitions indicated as in (B).
Molecular Cell 1998 1, DOI: ( /S (00) )

4. Figure 4
Secondary Structure Mediates Instability by Inhibiting DNA Polymerase during Replication
(A) Cycle sequencing of plasmids containing GAA repeats. (1) CTT62; (2) CTT250 using primer 1, which anneals 279 bases upstream of repeat (see Figure 4B); (3) CTT250 using primer 2, which anneals immediately upstream of the repeats (see [B]). Arrows indicate beginning and end of the repeat sequence-CTT. For CTT250, the end of the repeat region is not reached and is not marked with an arrow.
(B) Primer positions used in (A).
(C) In vitro replication by primer extension of single-stranded CTT template containing 36 repeats. Each reaction contained one dideoxy nucleotide (G, T, A, or C) for sequence information and to determine position of blocks. Control, polymerase alone; +SSB, bacterial single-stranded binding protein added; +RP-A, human RP-A added. Replication arrest is evident from very intense band, which can be seen in all lanes (e.g., Control). There is a block at repeat number 18 (n = 18). (n = 1, beginning of the repeats).
Molecular Cell 1998 1, DOI: ( /S (00) )

5. Figure 3
Instability of GAA/CTT Trinucleotides Occurs in Long but Not Short Repeats
(A) Gel-purified PCR fragment (575 repeats; arrow) amplified from a frataxin patient is the starting material for CTT 250.
(B) Repeat-containing fragments were gel purified and subcloned into pcDNA3(-).
(C) Instability assay for CTT or GAA repeats of 23, 42, 79, or 250. Groups of four bands represent input DNA (after 12 hr growth), and 1, 2, and 3 days of growth, except GAA 250 for which input DNA and 4 days are shown. For both CTT250 and GAA250, the process of subcloning and colony growth resulted in significant deletion (95% of the material had deleted to 250 repeats). The arrow indicates the position of the original, gel purified fragment (575 repeats).
Molecular Cell 1998 1, DOI: ( /S (00) )

6. Figure 5
A YRY Triplex Mediates Replication Arrest in Repeating GAA/CTT
(A) Base pairing in RRY and YRY triplexes. (Top) general models for RRY or YRY triplexes from GAA/CTT; (bottom) base pairs that would stabilize RRY or YRY triplexes. Note that YRY but not RRY is stabilized by C protonation. Both involve third-strand pairing to N7 of guanine.
(B) In vitro replication from single-stranded CTT8 (left) and double-stranded CTT250 (right). Note that for long repeats, the replication block is pH-sensitive.
Molecular Cell 1998 1, DOI: ( /S (00) )

7. Figure 6 Instability Occurs by an Intraallelic Mechanism in FRDA
(A) Experimental protocol. Offspring combinations from two heterozygous parents.
(B) Summary of rates of instability in long and short alleles (data from Figure 1) of offspring of heterozygous parents.
(C) Representative pedigree for (A).
(D) Experimental protocol. Homozygous or heterozygous parents transmitting a long allele to heterozygous offspring.
(E) Summary of the rate of instability in both the long and short alleles from the data in Figure 1 for heterozygous offspring.
(F) Representative pedigree for (D).
Molecular Cell 1998 1, DOI: ( /S (00) )

8. Figure 7 Common Model for Hairpin- and Triplex-Dependent Instability
Possible base pairing schemes for hairpin (CNG) and triplex (GAA). Structure may form on either unpaired strand. Repair of slippage on daughter (black) strand yields expansion; repair of slippage on template (red) strand causes deletion. Unpaired bases might facilitate intraallelic interactions with a sister chromatid or with a nearby lagging strand if structure forms on the leading strand. Dashes, base pairs; dots, nonstandard pairing.
Molecular Cell 1998 1, DOI: ( /S (00) )