Generation of Gross Chromosomal Rearrangements by a Single Engineered DNA Double Strand Break

Zhijun Qiu,Zhenhua Zhang,Anna Roschke,Peter D Aplan,Tamas Varga

doi:10.1038/srep43156

Abstract

Gross chromosomal rearrangements (GCRs), including translocations, inversions amplifications, and deletions, can be causal events leading to malignant transformation. GCRs are thought to be triggered by DNA double strand breaks (DSBs), which in turn can be spontaneous or induced by external agents (eg. cytotoxic chemotherapy, ionizing radiation). It has been shown that induction of DNA DSBs at two defined loci can produce stable balanced chromosomal translocations, however, a single engineered DNA DSB could not. Herein, we report that although a single engineered DNA DSB in H2AX “knockdown” cells did not generate GCRs, repair of a single engineered DNA DSB in fibroblasts that had ablated H2ax did produce clonal, stable GCRs, including balanced translocations and megabase-pair inversions. Upon correction of the H2ax deficiency, cells no longer generated GCRs following a single engineered DNA DSB. These findings demonstrate that clonal, stable GCRs can be produced by a single engineered DNA DSB in H2ax knockout cells, and that the production of these GCRs is ameliorated by H2ax expression.

Highlights

Common mechanism implicated seems to be inappropriate repair of one or more DNA double strand breaks (DSBs) by either the classic or error-prone, alternative NHEJ pathways
In order to address questions such as which forms of Gross chromosomal rearrangements (GCRs) are most common in the absence of oncogenic selection for specific GCRs, and whether defects in DNA DSB repair pathways would lead to increased frequency of GCRs, again in the absence of oncogenic selection for specific GCRs, a number of investigators have developed experimental systems in which a single DNA DSB can be introduced into a mammalian genome[19,20]
We modify the experimental approach to determine if clonal, stable GCRs can be produced in cells that are deficient for DNA repair components, such as NHEJ proteins or H2ax, a variant histone which becomes phosphorylated in response to DNA DSB and “coats” the DNA in the region of the DNA DSB24

Summary

Introduction

Common mechanism implicated seems to be inappropriate repair of one or more DNA DSB by either the classic or error-prone, alternative NHEJ pathways. We were unable to detect any clonal, stable GCRs (GCRs that are stable and found in a clonal population of daughter cells), such as balanced chromosomal translocations or megabase chromosomal inversions, following the introduction of a single, I-SceI- engineered DNA DSB21,22. These findings were consistent with previous reports that the frequency of chromosomal translocations induced by a single DNA DSB in mouse embryonic stem (ES) cells was extraordinarily rare (

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Conclusion