A homologous recombination defect affects replication-fork progression in mammalian cells

Fayza Daboussi,Bernard S. Lopez,Patricia Kannouche,Sylvain Courbet,Michelle Debatisse,Simone Benhamou,Malgorzata Z. Zdzienicka

doi:10.1242/jcs.010330

Abstract

Faithful genome transmission requires a network of pathways coordinating DNA replication to DNA repair and recombination. Here, we used molecular combing to measure the impact of homologous recombination (HR) on the velocity of DNA replication forks. We used three hamster cell lines defective in HR either by overexpression of a RAD51 dominant-negative form, or by a defect in the RAD51 paralogue XRCC2 or the breast tumor suppressor BRCA2. Irrespectively of the type or extent of HR alteration, all three cell lines exhibited a similar reduction in the rate of replication-fork progression, associated with an increase in the density of replication forks. Importantly, this phenotype was completely reversed in complemented derivatives of Xrcc2 and Brca2 mutants. These data reveal a novel role for HR, different from the reactivation of stalled replication forks, which may play an important role in genome stability and thus in tumor protection.

Highlights

Replication forks are routinely arrested by a broad variety of stresses (Hyrien, 2000; Shechter and Gautier, 2004)
Treatment with cisplatin reduces replication kinetics, an effect that is abrogated by mutation of the homologous recombination (HR) gene Xrcc3 (HenryMowatt et al, 2003)
We studied complemented cell-lines derived from Xrcc2 and Brca2 mutant cell lines

Summary

Introduction

Replication forks are routinely arrested by a broad variety of stresses (Hyrien, 2000; Shechter and Gautier, 2004). The relationships between homologous recombination (HR) and DNA replication have been well documented in cells challenged with strong genotoxic stresses. HR reactivates replication forks arrested at DNA lesions (Kuzminov, 1995). In mammalian cells, prolonged inhibition of replication progression generates DNA double-strand breaks and stimulates HR (Saintigny et al, 2001). The pivotal HR protein RAD51 localizes at arrested replication forks (Sengupta et al, 2003; Sengupta et al, 2004). Treatment with cisplatin reduces replication kinetics, an effect that is abrogated by mutation of the HR gene Xrcc (HenryMowatt et al, 2003)

Methods

Results

Conclusion