title: 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.
authors.affiliation: Department of Molecular and Human Genetics , Baylor College of Medicine , Houston , United States ; Department of Molecular Virology and Microbiology , Baylor College of Medicine , Houston , United States ; Dan L Duncan Cancer Center, Baylor College of Medicine , Houston , United States ; Department of Biochemistry, Molecular Biology , Baylor College of Medicine , Houston , United States.
authors.forename: Chandan
authors.initials: C
authors.lastname: Shee
authors.forename: Ben D
authors.initials: BD
authors.lastname: Cox
authors.forename: Franklin
authors.initials: F
authors.lastname: Gu
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Luengas
authors.forename: Mohan C
authors.initials: MC
authors.lastname: Joshi
authors.forename: Li-Ya
authors.initials: LY
authors.lastname: Chiu
authors.forename: David
authors.initials: D
authors.lastname: Magnan
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Halliday
authors.forename: Ryan L
authors.initials: RL
authors.lastname: Frisch
authors.forename: Janet L
authors.initials: JL
authors.lastname: Gibson
authors.forename: Ralf Bernd
authors.initials: RB
authors.lastname: Nehring
authors.forename: Huong G
authors.initials: HG
authors.lastname: Do
authors.forename: Marcos
authors.initials: M
authors.lastname: Hernandez
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Christophe
authors.initials: C
authors.lastname: Herman
authors.forename: P J
authors.initials: PJ
authors.lastname: Hastings
authors.forename: David
authors.initials: D
authors.lastname: Bates
authors.forename: Reuben S
authors.initials: RS
authors.lastname: Harris
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Miller
authors.forename: Susan M
authors.initials: SM
authors.lastname: Rosenberg
mesh_terms: D000818:Animals; D010583:Bacteriophage mu / Q000737:chemistry; D002876:Chromosomes, Bacterial / Q000737:chemistry / Q000378:metabolism*; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D000072200:Ku Autoantigen; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D058615:Synthetic Biology; D000071857:Tumor Suppressor p53-Binding Protein 1; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D014764:Viral Proteins; C065410:gam protein, Coliphage; D049452:Green Fluorescent Proteins; D004247:DNA; C121399:APOBEC3A protein, human; D003564:Cytidine Deaminase; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen
keywords: DNA double-strand breaks; E. coli; GFP; Human; Mouse; endogenous DNA damage; fluorescent-protein fusions; spontaneous DNA breaks; synthetic biology