Engineered proteins detect spontaneous DNA breakage in human and bacterial cells
- Baylor College of Medicine, United States
- Dan L Duncan Cancer Center, Baylor College of Medicine, United States
- University of Texas, United States
- University of Minnesota, United States
- the University of Texas MD Anderson Cancer Center, United States
Figures
Figure 1 with 2 supplements
GamGFP production mimics recB double-strand-exonuclease defect.
(A) Doxycycline-inducible gam-gfp fusion construct in the E. coli chromosome. Constitutively produced TetR protein represses the PN25tetO promoter, which produces GamGFP upon doxycycline induction. o…
Figure 1—figure supplement 1
Production of Gam and GamGFP fusion proteins in E. coli.
Doxycycline induction and detection of plasmid-borne Gam and chromosomally encoded GamGFP and GFP are performed by Coomassie blue staining following electrophoresis (Gam) or by western blot …
Figure 1—figure supplement 2
Long-term GamGFP production reduces E. coli viability.
Greater viability loss with GamGFP than Gam implies that GamGFP is a superior DSE trap. (A) We quantified the effect of long-term Gam and GamGFP production on cell viability by inducing Gam or …
Figure 2 with 2 supplements
GamGFP foci at DSBs in living E. coli.
(A) Strategy. In log-phase replicating E. coli, cells have more copies of origin (oriC)-proximal than terminus (ter)-proximal DNA and so will have more DSBs per cell when cleaved by chromosomally …
Figure 2—Figure supplement 1
Quantitative real-time PCR shows ∼three-fold more DNA copies near ori than ter in log-phase, regardless of I-SceI cleavage, implying that some cells have two and some have four ori:ter regions.
https://doi.org/10.7554/eLife.01222.007
Figure 2—figure supplement 2
Linear gamma-ray dose-response and bleomycin induction of GamGFP foci in E. coli.
(A) Numbers of GamGFP foci are linearly correlated with dose of DSB-producing γ-irradiation. Numbers of foci at different doses of γ-irradiation were calculated from the data displayed in Figure 2F. …
Figure 3
Subcellular/subgenomic localization of DSBs in living E. coli.
(A) Strategy: we varied the location of I-SceI cleavage sites (I-sites) in different strains relative to a fixed-position chromosomal TetR-mCherry-bound tetO array, with GamGFP temperature inducibly …
Figure 4 with 1 supplement
Generation-dependence of spontaneous GamGFP focus formation in proliferating E. coli.
Log-phase GamGFP-pre-induced cells were loaded into a microfluidic chamber in which single cells anchor then divide to form single-cell-layer microcolonies. The numbers of cell divisions and …
Figure 4—Figure supplement 1
Representative data on the origins of spontaneous DSBs over time during growth, or growth retardation, visualized and quantified per ‘Materials and methods’, Microfluidics and time-lapse fluorescence microscopy of E. coli.
Numbers in the lower right of each frame are hours after loading into the microfluidic chamber. GamGFP foci are indicated with arrows. Note that cells expressing gam-gfp show variation in the amount …
Figure 5 with 2 supplements
GamGFP marks DSBs in mammalian cells and is inhibited by Ku.
(A) GamGFP co-localizes with 53BP1 on laser-induced DNA breaks. (B) Ku inhibits recruitment of GamGFP to laser-induced damage, live cells. (C and D) Ku inhibits recruitment of GamGFP, fixed cells. …
Figure 5—figure supplement 1
GamGFP marks DSBs in mammalian cells.
(A) Live analysis of GamGFP localization to laser-induced DNA damage. Hela cells producing GamGFP were laser damaged along the cell track indicated by the red line at 0 min (m) and images were taken …
Figure 5—Figure supplement 2
Ku inhibits GamGFP recruitment at DSBs independently of non-homologous end joining.
Cells lacking either Ku80 or LigIV are defective in non-homologous end joining (NHEJ), yet the presence of Ku still inhibits recruitment of GamGFP to laser-induced DSBs even in NHEJ-defective cells, …
Figure 6
GamGFP inhibits IR-induced RAD51 foci, apparently blocking end resection.
We quantified RAD51 foci (single-stranded DNA) (Raderschall et al., 1999) induced by IR, so presumably at DSBs, in S-G2 (CyclinA-positive) cells that either did or did not produce GamGFP, from the …
Figure 7
APOBEC3A induces DSBs in human cells.
(A) HeLa cells co-transfected with GamEmGFP and APOBEC3A-mCherry or catalytic mutant, APOBEC3A-E72A-mCherry. (B) Summary of foci observed in cells producing both GamEmGFP and A3A-mCherry or …
Figure 8
Spontaneous DNA breakage in G1-phase cells: GamGFP shows large spontaneous G1 53BP1 foci to contain multi-break clusters.
The large spontaneous 53BP1 foci in undamaged cells, which occur solely in G1 (Harrigan et al., 2011; Lukas et al., 2011a), contain multiple DSBs that are marked by GamGFP. The GamGFP-53BP1 …
Additional files
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Supplementary file 1
(A) Escherichia coli K12 plasmids and strains used in this study. E. coli strains and plasmids were constructed using standard P1 transduction (Miller, 1992), transformation, lysogenization (Gumbiner-Russo et al., 2001), recombinant DNA (Sambrook and Russell, 2001) and phage λ Red-mediated short-homology recombineering methods (Datsenko and Wanner, 2000). Sequencing was performed by SeqWright DNA Technology Services (Houston, TX). (B) PCR primers.
- https://doi.org/10.7554/eLife.01222.018
