No Evidence for “Break-Induced Replication” in a Higher Plant – But Break-Induced Conversion May Occur

Veit Schubert,Ingo Schubert,Jörg Fuchs

doi:10.3389/fpls.2011.00008

Abstract

“Break-induced replication” (BIR) is considered as one way to repair DNA double-strand breaks (DSBs). BIR is defined as replication of the proximal break-ends up to the end of the broken chromosome using an undamaged (homologous) double-stranded template and mimicking a non-reciprocal translocation. This phenomenon was detected by genetic experiments in yeast. BIR is assumed to occur also in mammals, but experimental evidence is not yet at hand. We have studied chromosomes of the field bean, Vicia faba L., with respect to the occurrence of BIR after DSB induction during S and G2 phase. Simultaneous incorporation of the base analog ethynyldeoxyuridine (EdU) revealed no chromosomal replication pattern indicative of BIR. Thus, if occurring at all, BIR does not play a major role in DSB repair in higher plants with large chromosome arms. However, the frequency of interstitial asymmetric EdU incorporation within heterochromatic regions, visible on metaphase chromosomes, increased after chromosome breakage during S and G2 phase. Such asymmetric labeling could be interpreted as conservative replication up to the next replicon, circumventing a DSB, and yielding an interstitial conversion-like event.

Highlights

Double-strand breaks (DSBs) are the most critical DNA lesions
Of the chromatid aberrations observed after bleomycin and bleomycin plus EdU treatment during S and G2, reciprocal translocations (25), isochromatid breaks (36), and interstitial deletions (7) require two DSBs each, while simple chromatid breaks (87) representing terminal deletions, go back to a single DSB
Considering that a fraction of DSBs might have been repaired via pathways not resulting in microscopically detectable chromatid-type aberrations, the actual number of DSBs may be higher

Summary

Introduction

Double-strand breaks (DSBs) are the most critical DNA lesions. Unrepaired DSBs are usually lethal for dividing cells due to the subsequent loss of the acentric fragment and the unstable breakend of the centric fragment. As soon as overhanging ends meet at short stretches of complementary bases (single-strand annealing, SSA), the distal free ends are resected, and the break becomes ligated, resulting in a (micro-)deletion (Figure 1A). The break-ends may invade undamaged homologous template strands and a limited DNA synthesis across the break region is followed by re-ligation of both strands (synthesis-dependent strand annealing, SDSA). The result is genetically not detectable when the template was the undamaged sister chromatid; it appears as a gene conversion if an allele of the homologous chromosome served as a template (Figure 1C). When the Holiday junction (caused by break-end invasion into a template double strand) is resolved in connection with an exchange of the flanking region (Figure 1C), depending on the template double helix involved, a sister chromatid exchange, a crossing over, or a reciprocal translocation results

Methods

Results

Conclusion