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First published online 18 December 2007
doi: 10.1242/jcs.010330

Journal of Cell Science 121, 162-166 (2008)
Published by The Company of Biologists 2008
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A homologous recombination defect affects replication-fork progression in mammalian cells

Fayza Daboussi1, Sylvain Courbet2, Simone Benhamou3, Patricia Kannouche3, Malgorzata Z. Zdzienicka4, Michelle Debatisse2 and Bernard S. Lopez1,*

1 UMR 217 CNRS, Institut de Radiobiologie Cellulaire et Moléculaire, 18 route du panorama, 92265, Fontenay aux Roses, Cédex, France
2 UMR 7147 CNRS/Institut Curie, 26 rue d'Ulm, 75 248, Paris Cédex 05, France
3 FRE 2939, Institut Gustave Roussy, 94800, Villejuif, France
4 Department of Molecular Cell Genetics, Nicolaus-Copernicus-University in Torun, ul. Sklodowskiej-Curie 9, 85-094 Bydgoszcz, Poland


Figure 1
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  		Fig. 1. Analysis of replication elongation by molecular combing. (A) Example of labeled molecules with symmetric and asymmetric replication signals. Blue, anti-DNA; green, anti-IdU; red, anti-CldU. (B) Typical examples of labeled molecules obtained from HR proficient (upper panel) or HR-deficient cells (lower panel). (C) Percentage of forks traveling at the indicated speed (kb/minute) in the three HR-deficient cell lines, their respective parental cell line and complemented cell line derivatives. The analysis was performed on 150 DNA fibers with symmetric labeling. The dotted line corresponds to the mean rate in parental V79 or V79-puro cells. We compared the distributions of V79SM, Xrcc2-defective and Xrcc2-complemented cells with the distribution of V79. Consistently, there were no statistical differences between the complemented and the wild-type parental cell lines (P=0.5 for Xrcc2 and P=0.42 for Brca2). By contrast, the distributions between mutant and parental V79 or V79-puro or complemented derivatives were significantly different (P<0.001). There is no statistical difference between the complemented and parental cell lines (P=0.5 for Xrcc2 and P=0.42 for Brca2).

 

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  		Fig. 2. FACS analysis of S-phase progression. DNA synthesis measured by BrdU incorporation. Graphs are representative of eight independent experiments. The mean BrdU fluorescence intensity and the percentage of cells in S phase are indicated for each cell line. The apparent increase in S-phase cells in HR-defective cells was not statistically significant.

 

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© The Company of Biologists Ltd 2008