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Replication dynamics at common fragile site FRA6E

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Abstract

The replication dynamics at common fragile site FRA6E has been evaluated by molecular combing and interphase fluorescent in situ hybridisation (FISH) in primary human lymphocytes cultured under normal or aphidicolin-induced stress conditions. FRA6E is one of the most frequently expressed common fragile sites of the human genome. It harbours several genes, PARK2 being regarded as the most relevant one. According to the results obtained from interphase FISH analysis, FRA6E can be considered a mid-late-replicating sequence characterised by heterogeneous replication timing. Molecular combing did not reveal specific replication parameters at the fragile site: fork rates were highly comparable to those detected at an early replicating locus (LMNB2) used as control and in very good agreement with the whole-genome data obtained in parallel. The same indication applied to the density of initiation zones, the inter-origin distances from adjacent ongoing forks, the frequencies of unidirectional forks, fork arrest events and asynchronous forks. Interestingly, PARK2 appeared embedded in an early/late replication transition zone, corresponding to intron 8 (162 kb) and to the fragility core of FRA6E. In cells exposed to aphidicolin, few forks progressing at a rather slow rate were observed, the majority of them being unidirectional, but again a specific response of the fragile site was not observed. In summary, at FRA6E the replication process is not impaired per se, but chromosome breakages occur preferentially at an early/late replication transition zone. Aphidicolin might increase the occurrence of breakage events at FRA6E by prolonging the time interval separating the replication of early and late replication domains. These results may be of general significance to address the problem of fragile site instability.

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Acknowledgments

We are grateful to V. Bianchi (Department of Biology, University of Padova, Italy) and F. Pacchierotti (ENEA, CR Casaccia, Italy) for critical reading of the manuscript. This work was granted by the University of Padova (CPDA054385). A.R. has been the recipient of short-term travel grants from UICC (year 2004), EMBO (year 2005) and Ville de Paris (year 2006), which allowed her to learn the DNA molecular combing at the Institute Pasteur of Paris (France) in the former laboratory of A.B.

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  1. Department of Biology, University of Padova, Via U. Bassi 58/b, 35131, Padova, Italy

    Elisa Palumbo, Laura Matricardi, Elena Tosoni & Antonella Russo

  2. Genomic Vision, 29 rue du Faubourg Saint Jacques, 75014, Paris, France

    Aaron Bensimon

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  1. Elisa Palumbo
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  2. Laura Matricardi
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Correspondence to Antonella Russo.

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Communicated by E. Nigg

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Online resource 1

Schematic representation of FRA6E-PARK2 (A) and LAMIN B2 (B) genomic regions. Genes are represented by brownish-red bars, and those of interest are highlighted in purple. The genomic clones used as probes in FISH experiments on combed DNA are shown as blue bars. Probe RP11-21107, used only in interphase FISH experiments, is not visible, as it maps in 6q at 163.12-163.14 Mb. The position of flexibility peaks (detected as described in Online Resource 3) is indicated by a black line at the bottom of each panel. Clusters of flexibility are represented by green bars. Adapted from Ensembl genome browser. (GIF 70 kb)

High resolution image file (TIFF 1035 kb)

Online resource 2

Flow cytometric analysis of APH treated lymphocytes. (GIF 90 kb)

High resolution image file (TIFF 368 kb)

Online resource 3

(DOC 21 kb)

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Palumbo, E., Matricardi, L., Tosoni, E. et al. Replication dynamics at common fragile site FRA6E . Chromosoma 119, 575–587 (2010). https://doi.org/10.1007/s00412-010-0279-4

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