Fig 1.
Replication timing of mutant and normal FXN alleles.
Percentages are calculated from pooled data obtained with FRDA (GM15850 and GM16227) and control GM15851 cells. Per each group, at least 550 nuclei were analyzed from at least two independent replicated experiments (Raw data in S1 Table). SS = nuclei with two single FISH spots (non-replicated alleles); SD = nuclei with one single and one duplicated FISH signal (one allele has been replicated); DD = nuclei with two duplicated FISH signals (both alleles have been replicated); others = nuclei with one or none FISH signals. Error bars indicate standard errors of proportions. The probe used in these experiments is BAC RP11-265B8. For comparison, the replication timing of a late replication sequence (FRA3B, probe RP11-468L11) in normal GM15851 cells is shown. Examples of FISH replication patterns are shown in the bottom of the Figure.
Table 1.
Replication timing of normal and mutant FXN alleles according to interphase FISH after FACS cell sorting.
Fig 2.
Distributions of fork rates and Inter-Origin Distances (IOD) in a 850 kb region centred around FXN.
(A) Fork rate distributions observed in two normal (GM15851 and H691) and two FRDA (GM15850 and GM16227) cell lines. (B) IOD distributions observed in the same cell lines as above. No significant differences were detected for both variables described (Kruskal-Wallis non-parametric test).
Table 2.
Replication profile of the 850 kb genomic region harboring Frataxin.
Fig 3.
Synoptical view of the initiation events observed in a 850 kb region surrounding FXN.
Top: the region investigated in the study. Green lines represent the three BAC clones used as probes, gray lines display genes, FXN is highlighted in red. The positions of primer sets used in the Short Nascent Strand (SNS) abundance assay are indicated in the map. In the four following schemes, blue arrows indicate the position of each bidirectional origin identified within normal (GM15851, H691) and mutant (GM15850, GM16227) alleles, relatively to the three probes (green) and the FXN gene (red). The position of the GAA-repeat expansion in the mutated alleles is also displayed.
Fig 4.
Two representative images of the FXN locus as detected by FISH and molecular combing.
Three probes (green) are used to detect the 850 kb region harboring FXN (red). The position of the GAA-repeat expansion in the mutated alleles is also displayed. Replication tracks are visualized in blue (IdU) and red (CldU), arrows indicate origin positions, the asterisk corresponds to a paused/arrested fork. The two images refer to S8 Fig, molecules 41 and 5 respectively. The region identified by BAC RP11-265B8, where FXN maps, is enlarged to allow a better visualization of origin firing and a sharp interpretation of the replication signals. For each enlargement, the first frame corresponds to the merged image, the second frame shows the probes in green fluorescence, the third one displays the blue and red tracks coinciding with the replicative patterns. Calibration bar = 100 kb.
Fig 5.
Position, direction and extension of the replication forks with unidirectional pattern observed in the genomic region under study.
(A) The region investigated with green lines representing the three BAC clones used as probes, gray lines displaying genes, FXN (highlighted in red). In the following schemes, unidirectional fork progression within normal (GM15851, H691) and mutated (GM15850, GM16227) alleles is represented by blue arrows relatively to the three probes (green) and the FXN gene (red). The position of the GAA-repeat expansion in the mutated alleles is also displayed. (B) Length distributions of the unidirectional running forks observed in normal and mutant cell lines, in the region including the central BAC and the flanking probe-to-probe distances. There is a significant length reduction in FRDA cells with respect to normal ones (P < 0.01, Kruskal-Wallis non-parametric test).
Fig 6.
Synoptical view of the events of pause/arrest of the replication fork recorded in the genomic region under study.
The region investigated is shown on the top: green lines represent the three BAC clones used as probes, gray lines display genes, FXN is highlighted in red. In the four following schemes, each red asterisk indicates a block in fork progression within normal (GM15851, H691) and mutated (GM15850, GM16227) alleles, relatively to the three probes (green) and the FXN gene (red). The position of the GAA-repeat expansion in the mutated alleles is also displayed.
Fig 7.
(A) The map position of primer sets used in the Short Nascent Strand (SNS) abundance assay. Green lines represent the three BAC clones used as probes in molecular combing analyses; gray lines display genes, FXN is highlighted in red. (B) Abundance of FXN sequences in nascent DNA from two independent isolation experiments for each cell line. The mean quantities of SNS at FXN region (primers F1-F4) were normalized to the average quantities determined in the control sites on chr. 9 (primers C1-C3). (C) Abundance of SNS at each primer set on chr. 9 was normalized to LB2C1, and further corrected according to the enrichment at the LAMIN B2 origin, which was set as threshold. (D) Abundance of SNS at each primer set on chr. 9 is shown as pooled data for FRDA (GM16227, GM15850) and control cells (GM15851 and H691); the analysis was performed as in C. Error bars are standard errors of the mean.