Tammaro M et al. (2016), DNA double-strand breaks with 5' adducts are ef...

XB-ART-51275

Nucleic Acids Res 2016 Jan 08;441:221-31. doi: 10.1093/nar/gkv969.

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DNA double-strand breaks with 5' adducts are efficiently channeled to the DNA2-mediated resection pathway.

Tammaro M , Liao S , Beeharry N , Yan H .

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DNA double-strand breaks (DSBs) with 5' adducts are frequently formed from many nucleic acid processing enzymes, in particular DNA topoisomerase 2 (TOP2). The key intermediate of TOP2 catalysis is the covalent complex (TOP2cc), consisting of two TOP2 subunits covalently linked to the 5' ends of the nicked DNA. In cells, TOP2ccs can be trapped by cancer drugs such as etoposide and then converted into DNA double-strand breaks (DSBs) that carry adducts at the 5' end. The repair of such DSBs is critical to the survival of cells, but the underlying mechanism is still not well understood. We found that etoposide-induced DSBs are efficiently resected into 3' single-stranded DNA in cells and the major nuclease for resection is the DNA2 protein. DNA substrates carrying model 5' adducts were efficiently resected in Xenopus egg extracts and immunodepletion of Xenopus DNA2 also strongly inhibited resection. These results suggest that DNA2-mediated resection is a major mechanism for the repair of DSBs with 5' adducts.

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Species referenced: Xenopus
Genes referenced: avd cenpf dna2 exo1 rpa1 top2a

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	Figure 1. Etoposide-induced RPA foci represent 3′ ss-DNA resected from DSBs. (A) Etoposide but not ICRF193 induces RPA foci in U2OS cells. U2OS cells were treated with 250 μM etoposide or ICRF193 for 2 h, fixed, and stained for RPA and CenpF. (B) Etoposide-induced RPA foci are sensitive to 3′->5′ ss-DNA exonuclease. U2OS cells were treated with 250 μM etoposide for 2 h, permeabilized, incubated with RecJ (5′->3′ ss-exonuclease) or E.coli Exo1 (3′->5′ ss-exonuclease) for 2 h, and finally fixed and stained for RPA.
	Figure 2. Effect of DNA2 siRNA on cell proliferation. U2OS cells were treated with two rounds of 20 nM control or DNA2 siRNA for 72 h and then subjected to various analyses. (A) Western blot of DNA2. (B) Western blot of other resection proteins. (C) Immunofluorescence staining of the siRNA-treated cells exposed to ETP. (D) Plots of the percentages of EdU+ cells and CenpF+ cells. Over 100 nuclei were counted and the percentages from three experiments were used for calculations of the averages and standard deviations.
	Figure 3. DNA2 siRNA causes a partial inhibition of RPA focus induction by etoposide. Cells were treated with two rounds of 20 nM control siRNA (A) or DNA2 siRNA (B) for 72 h, exposed to 250 μM etoposide for 2 h and finally fixed and stained for RPA, EdU and CenpF. (C) Close-ups of the nuclei indicated in (A) and (B). (D) The percentages of CenpF+ cells with etoposide-induced RPA foci were quantified and plotted. Over 100 nuclei were counted and the percentages from nine experiments were used for calculations of the averages and standard deviations.
	Figure 4. Complementation of DNA2 siRNA's effect by the siRNA-resistant DNA2 gene. U2OS cells (A) and U2OS cells expressing the siRNA-resistant DNA2 gene (B) were treated with two rounds of 20 nM DNA2 siRNA for a total of 72 h, then exposed to 250 μM etoposide and finally fixed and stained for RPA, EdU and CenpF. (C) Close-ups of the nuclei indicated in (A) and (B). (D) The percentages of CenpF+ cells with etoposide-induced RPA foci were quantified and plotted. Over 100 nuclei were counted and the percentages from three experiments were used for calculations of the averages and standard deviations.
	Figure 5. U2OS cells partially depleted of DNA2 are hyper-sensitive to etoposide. U2OS cells treated with 1 round of 10 nM control or DNA2 siRNA for 48 h were exposed to etoposide at the indicated concentrations for 2 h and then allowed to grow in fresh media for 9 more days. (A) The resulting colonies were stained with crystal violet. (B) The percentages of colonies relative to the no drug well were quantified and plotted. The averages and standard deviations were calculated from the data of three experiments.
	Figure 6. Depletion of DNA2 inhibits resection of DNA with 5′ adducts. (A) DNA substrates bearing different types of ends were incubated at 1.5 ng/μl with the control or DNA2-depleted extracts. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.
	Figure 7. Rescue of the DNA2 depletion defect by the purified DNA2 protein. DNA substrates bearing different types of ends were incubated with the DNA2-depleted extracts supplemented with either buffer or DNA2 protein. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.
	Figure 8. DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

References [+] :

Alchanati, The E3 ubiquitin-ligase Bmi1/Ring1A controls the proteasomal degradation of Top2alpha cleavage complex - a potentially new drug target. 2009, Pubmed