DNA Repair Biosensor-Identified DNA Damage Activities of Endophyte Extracts from Garcinia cowa

Abstract

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

Keywords: DNA damage and repair; Garcinia cowa Roxb. ex Choisy; biosensor; cancer; endophyte.

Figure 1

Construction of fluorescent biosensor clones. (a) mCherry was inserted at the C-terminus of 53BP1. The non-targeted and targeted knockin of 53BP1 when the genomic DNA was cut with XbaI were 3.6 and 5.4 kbp. (b) For RAD54L, the non-targeted and targeted knockin when the genomic DNA was cut with SpeI and KpnI were 6.4 and 10 kbp. Codon regions are represented by boxes, and the gray-colored box is the last codon in each gene. The black triangle is the restriction site, and an open triangle denotes the stop codon. (c) Southern blotting of XbaI-digested (53BP1) and SpeI-KpnI-digested (RAD54L) genomic DNA. Definitions: mCherry, monomeric Cherry; mVenus, monomeric Venus; kbp, kilobase pair; 53BP1, tumor protein p53 binding protein 1; RAD54L, DNA repair protein RAD54-like; WT, wild type.

Figure 1

Construction of fluorescent biosensor clones. (a) mCherry was inserted at the C-terminus of 53BP1. The non-targeted and targeted knockin of 53BP1 when the genomic DNA was cut with XbaI were 3.6 and 5.4 kbp. (b) For RAD54L, the non-targeted and targeted knockin when the genomic DNA was cut with SpeI and KpnI were 6.4 and 10 kbp. Codon regions are represented by boxes, and the gray-colored box is the last codon in each gene. The black triangle is the restriction site, and an open triangle denotes the stop codon. (c) Southern blotting of XbaI-digested (53BP1) and SpeI-KpnI-digested (RAD54L) genomic DNA. Definitions: mCherry, monomeric Cherry; mVenus, monomeric Venus; kbp, kilobase pair; 53BP1, tumor protein p53 binding protein 1; RAD54L, DNA repair protein RAD54-like; WT, wild type.

Figure 2

Cell proliferation of biosensor clones was slightly lower than wild-type DT40. Two clones of targeted knockin were generated and they demonstrated the same phenotype. Data are shown as the mean ± standard deviation of three biologically independent experiments. Definitions: 53BP1-mCherry-RAD54L-mVenus#C1, tumor protein p53 binding protein-monomeric Cherry-RAD54-like-monomeric Venus knockin clone 1; 53BP1-mCherry-RAD54L-mVenus#C2, tumor protein p53 binding protein-monomeric Cherry-RAD54-like-monomeric Venus knockin clone 2; 53BP1, tumor protein p53 binding protein 1; RAD54L, DNA repair protein RAD54-like.

Figure 3

The viability of biosensor clones following exposure to DNA-damaging agents demonstrated the same phenotype as wild-type DT40. The experiments were reproduced with biologically independent replicates. Data are shown as the mean ± standard deviation from one experiment. The error bars are shorter than the height of the symbol in some points. Definitions: ETP, etoposide; CPT, camptothecin.

Figure 4

Foci formation of biosensor clones revealed the same phenotype as that of wild-type DT40. (a) γH2AX (red dot) focus formation of wild-type and biosensor cells when treated with either CPT or ETP. (b) Colocalization (yellow) of γH2AX (red) and 53BP1-mCherry (green) in wild-type and biosensor cells when treated with either CPT or ETP. (c) Colocalization (yellow) of 53BP1 antibody (red) and 53BP1-mCherry (green) in wild-type and biosensor cells when treated with ETP. (d) Colocalization (yellow) of the RAD51 antibody (red) and 53BP1-mCherry (green) in wild-type and biosensor cells when treated with CPT. The bar graphs are shown as the mean ± standard deviation. Definitions: 53BP1-mCherry-RAD54L-mVenus, tumor protein p53 binding protein-monomeric Cherry-RAD54-like-monomeric Venus knockin cells; WT, wild type, ETP, etoposide; CPT, camptothecin; Ab, antibody. Scale bar represents 10 μM.

Figure 4

Foci formation of biosensor clones revealed the same phenotype as that of wild-type DT40. (a) γH2AX (red dot) focus formation of wild-type and biosensor cells when treated with either CPT or ETP. (b) Colocalization (yellow) of γH2AX (red) and 53BP1-mCherry (green) in wild-type and biosensor cells when treated with either CPT or ETP. (c) Colocalization (yellow) of 53BP1 antibody (red) and 53BP1-mCherry (green) in wild-type and biosensor cells when treated with ETP. (d) Colocalization (yellow) of the RAD51 antibody (red) and 53BP1-mCherry (green) in wild-type and biosensor cells when treated with CPT. The bar graphs are shown as the mean ± standard deviation. Definitions: 53BP1-mCherry-RAD54L-mVenus, tumor protein p53 binding protein-monomeric Cherry-RAD54-like-monomeric Venus knockin cells; WT, wild type, ETP, etoposide; CPT, camptothecin; Ab, antibody. Scale bar represents 10 μM.

Figure 4

Foci formation of biosensor clones revealed the same phenotype as that of wild-type DT40. (a) γH2AX (red dot) focus formation of wild-type and biosensor cells when treated with either CPT or ETP. (b) Colocalization (yellow) of γH2AX (red) and 53BP1-mCherry (green) in wild-type and biosensor cells when treated with either CPT or ETP. (c) Colocalization (yellow) of 53BP1 antibody (red) and 53BP1-mCherry (green) in wild-type and biosensor cells when treated with ETP. (d) Colocalization (yellow) of the RAD51 antibody (red) and 53BP1-mCherry (green) in wild-type and biosensor cells when treated with CPT. The bar graphs are shown as the mean ± standard deviation. Definitions: 53BP1-mCherry-RAD54L-mVenus, tumor protein p53 binding protein-monomeric Cherry-RAD54-like-monomeric Venus knockin cells; WT, wild type, ETP, etoposide; CPT, camptothecin; Ab, antibody. Scale bar represents 10 μM.

Figure 5

Ethyl acetate crude extracts from Garcinia cowa Roxb. ex Choisy affected the biosensor cell viability. The viability assay of the biosensor cells was measured following exposure with either Garcinia cowa Roxb. ex Choisy ethyl acetate or ethanol crude extract for 48 h. The experiment was repeated with biologically independent replicates. Each point is shown as the mean ± standard deviation from one experiment.

Figure 6

Overall endophytic fungi isolation. (a) Garcinia cowa Roxb. ex Choisy leaves. (b) Morphology of Phyllosticta sp. on half strength potato dextrose agar (PDA) and (c) yeast malt agar (YMA). (d) Morphology of Phyllosticta sp. spore. The scale bar represents 20 µm.

Figure 7

Phyllosticta sp. YGE41 crude extracts affected the viability of biosensor cells. Thebiosensor cells were treated continuously with ethyl acetate crude metabolites from Phyllosticta sp. YGE41 for 48 h before viability determination. The viability assay was repeated with biologically independent replicates. Each point is shown as the mean ± standard deviation from one experiment.

Figure 8

Garcinia cowa and Phyllosticta sp. YGE41 crude extracts caused mild DSB and activated NHEJ. The biosensor cells were continuously treated with the crude extracts for 8 h and were collected at the indicated time. The bar graphs are shown as the mean ± standard deviation. The foci formation images are represented at 8 h. Definitions: DMSO, dimethyl sulfoxide; ETP, etoposide; CPT, camptothecin; ND, Student’s t-test cannot be determined.

Figure 9

Garcinia cowa and Phyllosticta sp. YGE41 crude extracts differentially affected NHEJ-deficient cells. The cells were treated continuously with ethyl acetate crude metabolites from Garcinia cowa Roxb. Ex Choisy and Phyllosticta sp. YGE41 to determine cell viability. The experiment was repeated with biologically independent replicates. Data are shown as the mean ± standard deviation. Student’s t-test was performed at 6.25, 12.5, 25, and 50 μg/mL of Garcinia cowa and Phyllosticta sp. YGE41. Definitions: DMSO, dimethyl sulfoxide; 53BP1^−/−, 53BP1 knockout; RAD54L^−/−, RAD54L knockout.