(A) The YAC breakage assay. Cells containing a YAC, which contains either the entire FRA16D region or subregions and the URA3 gene, are FOA^S. When breakage occurs inside the FRA16D region (heavy black line), the broken YAC can be rescued by the addition of a new telomere to the 108 bp C₄A₄ telomere seed sequence. The resulting cells will be Leu⁺, Trp⁺ and FOA^R. (B) YACs from the CEPH YAC library were modified by adding a telomere seed sequence and a LEU2 marker. Human sequences are represented by grey boxes (not to scale). The dark grey box represents the 270 kb FRA16D region defined as most fragile by (Reid et al., 2000). YAC 801B6 and YAC 972D3 are aligned according to their coordinates in the human genome. (C) The breakage assay was performed for YAC 801B6 and YAC 972D3. Bars represent the average of 3 experiments, SEM is shown. Statistical significance was determined using a pooled variant t-test, * P < 0.05. (D) Breakage intermediates initiate within FRA16D. Cells of the rad50Δ strain background containing the 801B6 YAC from the CEPH YAC library were arrested in G1, released into S phase, and samples collected every 30 min for 3 hours. Chromosomes were prepared and separated using PFGE, blotted, and hybridized to either a TRP1 or a URA3 probe to the left or right arm of the YACs, respectively. The endogenous chromosome IV (Containing TRP1) and V (containing URA3) and full length YAC 801B6 are indicated by long arrows. Initial breakage intermediates are indicated by asterisks (** or *), degradation products by black arrowheads, putative recombination products by grey arrowheads. (E) Mapping of the breakage intermediates, symbols as in (D). The diagram of YAC 801B6 is approximately to scale.

(A) Putative cruciform structure of Flex1(AT)34. (B) The secondary structure of the 314 bp fragment containing Flex1(AT)34 predicted by the Mfold Program (highest stability structure, Tm 60 °C). http://www.bioinfo.rpi.edu/applications/mfold/dna/ (Zuker, 2003).

FRA16D-dependent growth defect in the rad50Δ background. An equal amount cells containing either YAC 801B6 or YAC 972D3 were spotted on YC-Trp-Ura plates containing either no drug or 0.005 M or 0.01 M hydroxyurea. Three dilutions of 10⁻¹, 10⁻² and 10⁻³ are shown; the number of colonies in the second dilution is indicated. The top three panels represent one experiment; the bottom two panels represent a separate, independent experiment. Each panel shows colonies from one plate, all plates were incubated 3 days at 30 °C.

(A) Structure of the YACs containing the subregions of FRA16D diagrammed in (B) (denoted as “Flex”). Two types of YACs were created, either without (top) or with (bottom) the HIS5 gene. The black bars flanking the Flex region represent homologous sequences. For the four Flex1 sequences diagrammed, the dark grey bars represent the perfect AT repeat region. Flex1(AT)5, (AT)14 and (AT)23 are exactly the same except for the AT repeat number; Flex1(AT)34 contains 101 bp of extra distal sequence, and lacks 400 bp of the proximal sequences compared to the other Flex1 sequences. (B) The FlexStab score of the FRA16D region. Basepairs are on the X axis. Grey arrows represent the flexibility peaks used for this study. Below it, the exons of the WWOX/FOR gene are represented by grey boxes. Regions chosen for analysis in the present study are represented by black boxes: Flex4 is 1349 bp, Flex5-p is 2054 bp, Flex1 is 314–552 bp. The control sequence is 1320 bp or 400 bp. (C) The flexibility score of the right arm of the top YAC shown in (A). (D) Results of the breakage assay in the rad52Δ background using the top (URA3) YACs diagrammed in (A). The control is 1320 bp. The experiment was repeated at least 3 times for each strain, average with SEM is shown. * P < 0.05 compared to the control by a pooled variant t-test (E) Breakage assay using the Flex1-HIS5 YAC in the wild-type background in the absence (dark grey) or presence (light grey) of hydroxyurea. FOA^R colonies were replica plated to YC-His plates, and only the His⁻ colonies, indicating FOA^R due to YAC breakage, were used to calculate a rate of FOA^R His⁻. A 400 bp control was used (similar in size to the cloned Flex1 regions). The experiment was repeated at least 3 times for each strain, average with SEM is shown. * P < 0.05 compared to the respective control (either no drug control or 0.15 M hydroxyurea control) by a pooled variant t-test.

Flex1(AT)34 can stall replication fork progression. (A) Diagram of the plasmid containing the Flex1 regions used for 2D-gel analysis and predicted position on the Y arc of replication intermediates of a stall within Flex1 (circle). The 2μ origin replicates bi-directionally. (B) 2D-gel analysis of the replication intermediates traversing the 2914 bp XbaI fragment from plasmids replicated in yeast and containing the indicated Flex1 region. The AT perfect repeat is 191 bp from the second XbaI site for (AT)34 and 97 bp for (AT) 23, (AT)14 and (AT)5. A second experiment yielded the same results (not shown). (C) 2D gel analysis of replication intermediates isolated from cells grown in 0.1 M hydroxyurea (HU).

A Model for the Mechanism behind the Chromosome Breaks and Rearrangements Found at Common Fragile Sites. The type of secondary structure and exact location with respect to the stalled fork and broken chromosome are unknown, but are illustrated for diagrammatic purposes.