Piecing it together: New technique locates DNA breaks

Research Areas:

Genomics, Double-strand DNA breaks, Green fluorescent protein, Red fluorescent protein

Imaging Needs:

Protein co-localization

Imaging System:
  • Recombinant proteins expressing GFP and RFP
  • Zeiss Axio Imager Z1 microscope
  • Hamamatsu ImagEM EM-CCD camera
Measuring molecular events in living organisms

Co-localizing proteins at DNA breaks provides important insights into cellular biology. Recent techniques enable comparison of similar in vitro results with observations in living organisms. Learn more in Illuminating Activity—In Vitro and In Vivo. Read now.

THE QUESTION

How are double-strand breaks caused in genomic DNA?

Breakage of both strands in a DNA molecule creates a focus of genetic instability, and can cause gene deletions, chromosome loss, and point mutations. They have been implicated in genetic disease, including cancer, and in the development of antibiotic resistance in microbes.

THE BARRIERS

Double-strand breaks can be induced by radiation or endonuclases, but their frequency and causes in nature remain a mystery. Previous techniques to locate and quantify such breakages have suffered from poor sensitivity and low specificity for double-strand breaks versus single-strand breaks and nicks.

THE SOLUTION

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells
Shee C, Cox BD, Gu F, Luengas EM, Joshi MC, Chiu LY, Magnan D, Halliday JA, Frisch RL, Gibson JL, Nehring RB, Do HG, Hernandez M, Li L, Herman C, Hastings P, Bates D, Harris RS, Miller KM, Rosenberg SM
eLife. 2013 Oct 29(2): e01222. PMCID: PMC3809393.

Shee, et al,1 present a new method to detect and quantify double-strand DNA breaks using a viral protein called Gam tagged with green fluorescent protein (GFP). The recombinant GamGFP fusion protein binds to and prevents repair of double-strand breaks, enabling localization and quantification by fluorescence microscopy.

To demonstrate specifically of GamGFP protein for the ends of double-strand breaks, the authors created site-specific breaks using an endonuclease. Using a Hamamatsu ImagEM EM-CCD camera, they show that GamGFP co-localizes with an mCherry-tagged marker for the target DNA break.

The authors find that double-strand breakage rates in human and bacterial cells correspond with mitosis rates, indicating the breaks may be replication dependent. They also identify a double-strand cleavage enzyme that may be specific to primates. The GamGFP technique provides a precise and specific method to study double-strand DNA breakage in multiple species.

THE POSSIBILITIES

Shee, et al, relied on the low-light capabilities of Hamamatsu’s ImagEM EM-CCD technology to co-localize fluorescently tagged proteins at DNA breakage sites in vitro. Find out how Takeaki Ozawa and colleagues use an ImagEM EM-CCD camera to detect receptor binding in vivo using luminescence in place of fluorescence, in Illuminating Activity—in vitro and in vivo.

References

  1. Shee, et al. Engineered proteins detect spontaneous DNA breakage in human and bacterial cells. eLife. 2013 Oct 29(2): e01222. PMCID: PMC3809393.
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