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
Highlights
DNA double-strand breaks (DSBs) are the most genome-destabilizing DNA damage (Jackson and Bartek, 2009)
In human and mouse cells, we show that GamGFP labels DSBs and we provide evidence that— (i) GamGFP competes with Ku for DSBs; (ii) 53BP1 appears less specific for DSBs than GamGFP; (iii) GamGFP inhibits end resection at DSBs; (iv) DNA cytosine deamination produces DSBs in human cells, identifying a potentially primate-specific mechanism of DNA breakage; and (iv) G1 cells show multiple clustered foci, implying that some spontaneous DNA breakage occurs outside of S phase when most replication takes place
We show that chromosomally encoded Gam and GamGFP are functional in E. coli by demonstrating that their production blocks the action of RecBCD, a highly DSE-specific dsDNA exonuclease, in two assays
Summary
DNA double-strand breaks (DSBs) are the most genome-destabilizing DNA damage (Jackson and Bartek, 2009). ‘DSBs’ is used here as a collective term that includes two-ended structures (DSBs, e.g., as caused by double-strand endonucleases or ionizing radiation) and single double-stranded ends of DNA (DSEs, or one-ended DSBs), such as are caused by replication-fork collapses (Kuzminov, 2001). We use ‘DSE’ to refer to each single DSE in a two-ended DSB and to the sole DSE in a one-ended DSB. DSBs (one- and two-ended) promote deletions, genome rearrangements (Hastings et al, 2009), chromosome loss (Paques and Haber, 1999), and point mutations (Harris et al, 1994; Rosenberg et al, 1994; Strathern et al, 1995). DSB-induced genomic instability promotes cancer (Negrini et al, 2010) and genetic diseases (O’Driscoll and Jeggo, 2006), evolution of antibiotic resistance
Published Version (Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have