Kinetic analysis of DNA double-strand break repair pathways in Arabidopsis

Abstract

Double-strand breaks in genomic DNA (DSB) are potentially lethal lesions which separate parts of chromosome arms from their centromeres. Repair of DSB by recombination can generate mutations and further chromosomal rearrangements, making the regulation of recombination and the choice of recombination pathways of the highest importance. Although knowledge of recombination mechanisms has considerably advanced, the complex interrelationships and regulation of pathways are far from being fully understood.We analyse the different pathways of DSB repair acting in G2/M phase nuclei of irradiated plants, through quantitation of the kinetics of appearance and loss of γ-H2AX foci in Arabidopsis mutants. These analyses show the roles for the four major recombination pathways in post-S-phase DSB repair and that non-homologous recombination pathways constitute the major response. The data suggest a hierarchical organisation of DSB repair in these cells: C-NHEJ acts prior to B-NHEJ which can also inhibit MMEJ. Surprisingly the quadruple ku80 xrcc1 xrcc2 xpf mutant can repair DSB, although with severely altered kinetics. This repair leads to massive genetic instability with more than 50% of mitoses showing anaphase bridges following irradiation. This study thus clarifies the relationships between the different pathways of DSB repair in the living plant and points to the existence of novel DSB repair processes.