Results: 3

1.
Figure 3

Figure 3. From: DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.

Model for the contribution of NHEJ and HR to repair of DSBs during cell cycle. NHEJ is active during all stages of cell cycle, but its efficiency is the highest during G2/M. HR is active primarily in the S-phase and has lower activity in G2/M. The length of each cell cycle stage is proportional to the number of cells at a corresponding stage in unsynchronized culture of normal fibroblasts in .

Zhiyong Mao, et al. Cell Cycle. ;7(18):2902-2906.
2.
Figure 1

Figure 1. From: DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.

Reporter constructs for analysis of NHEJ and HR repair. (A) Reporter cassette for detection of NHEJ. The cassette consists of a GFP gene under a CMV promoter with an engineered intron from the rat Pem1 gene, interrupted by an adenoviral exon (Ad). The adenoviral exon is flanked by I-SceI recognition sites in inverted orientation (cell line I9a) or in direct orientation (cell line S13a) for induction of DSBs. In this construct the GFP gene is inactive; however upon induction of a DSB and successful NHEJ the construct becomes GFP+. SD, splice donor; SA, splice acceptor; shaded squares, polyadenylation sites. (B) Reporter cassette for detection of HR (cell lines H15c and H32c). The cassette consists of two mutated copies of GFP-Pem1. In the first copy of GFP-Pem1 the first GFP exon contains a deletion of 22 nt and an insertion of two I-SceI recognition sites in inverted orientation. The 22 nt deletion ensures that GFP cannot be reconstituted by a NHEJ event. The second copy of GFP-Pem1 lacks a promoter, the first ATG, and the second exon of GFP. Upon induction of DSBs by I-SceI, gene conversion events reconstitute an active GFP gene. (C) Incompatible DNA ends generated by digestion of two I-SceI sites in inverted orientation as in the lines NHEJ-I9a, HR-H15c, and HR-H32c. (D) Compatible DNA ends generated by digestion of two I-SceI sites in direct orientation as in the line NHEJ-S13a.

Zhiyong Mao, et al. Cell Cycle. ;7(18):2902-2906.
3.
Figure 2

Figure 2. From: DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.

DSB repair pathways during cell cycle. (A) Cell cycle distribution of HCA2-hTERT cells following indicated treatments. Number of cells is plotted against DNA content determined by PI staining. Cell cycle distribution was analyzed every day for 7 consecutive days. The images show confluent cells on day 7 after they reached confluence, and drug treated cells on day 4 after treatment, which is the time point when DSB repair took place. (B) Representative FACS traces for the analysis of NHEJ and HR. Cells were co-transfected with 5 µg of plasmid encoding I-SceI, and 0.1 µg of a DsRed plasmid, after they entered growth arrest as shown in panel A. Cells were analyzed by flow cytometry 4 days after transfection, using green-versus-red fluorescent plot as described previously . Green fluorescence is plotted on the x-axis and red fluorescence is plotted on the y-axis. The narrow triangular area on the diagonal corresponds to autofluorescent cells. Typically 20,000 cells were analyzed in each sample. In the treatments where the numbers of GFP+ cells were low, 40,000 cells were scored. (C, D) Frequency of NHEJ and HR at various cell cycle stages analyzed in two independent NHEJ and HR reporter cell lines. Each cell line contains a single integrated copy of an NHEJ or HR reporter cassette. The ratio of GFP+/DsRed+ cells is used as a measure of repair efficiency. NHEJ and HR are shown on different scales due to the large differences in repair frequency. The experiments were repeated at least four times and error bars are s. d. Stars (*) indicate the statistically significant differences (P<0.05, t-test).

Zhiyong Mao, et al. Cell Cycle. ;7(18):2902-2906.

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