Engineered proteins detect spontaneous DNA breakage in human and bacterial cells

Abstract

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells.

DOI: http://dx.doi.org/10.7554/eLife.01222.001

eLife digest

Cells have developed a variety of mechanisms for repairing DNA molecules when breaks occur in one or both of the DNA strands. However, we know relatively little about the causes of these breaks, which often occur naturally, or even about how common they are. Learning more about the most common forms of DNA breakage is important because the genomic changes caused by these breaks are driving forces behind both cancer and evolution, including the evolution of drug resistance in bacteria.

Shee et al. have developed a new method for detecting double-strand breaks in both bacterial and mammalian cells. The method involved combining a natural virus protein called Gam with a fluorescent protein called GFP (short for green fluorescent protein) to make a fusion protein called GamGFP. Gam was chosen because it binds only to double-strand breaks, traps double-strand breaks, and does not bind to any proteins. Genetic engineering techniques were used to introduce GamGFP into cells, with DNA breaks in these cells showing up as fluorescent spots when viewed under a microscope.

Shee et al. used this approach to detect double-strand breaks in both Escherichia coli cells and mammalian cells, and to measure the rate of spontaneous DNA breakage in E. coli. The number of double-strand breaks in E. coli was proportional to the number of times the cells had divided, which provides support for DNA replication-dependent models of spontaneous DNA breakage.

The GamGFP method also provided various insights into DNA breaks in mouse and human cells. In particular, Shee et al. found evidence for a mechanism of DNA breakage that appears to be specific to primates. This mechanism involves an enzyme that is only found in the innate immune system of primates removing an amine group from a cytosine. In future, this approach might allow the trapping, mapping and quantification of DNA breaks in all kinds of cells, and the highly specific way GamGFP binds to breaks could make it the preferred tool for studying DNA breakage in mammalian cells.

DOI: http://dx.doi.org/10.7554/eLife.01222.002