Endonuclease G: a role for the enzyme in recombination and cellular proliferation

Abstract

Our earlier studies had suggested that endonuclease G (EndoG), a member of the evolutionarily conserved DNA/RNA nonspecific betabetaalpha-Me-finger nuclease family, functioned in the a sequence-mediated segment inversion observed during herpes simplex virus 1 replication. To test this hypothesis, we used RNA interference to reduce the level of EndoG in mammalian cells in culture. Reduction of EndoG produced a small but statistically significant decrease in a sequence-mediated recombination, suggesting that EndoG does play a role in this process. We also observed that reduction in the level of EndoG resulted in a deficiency in cell proliferation. Cells with a reduced level of EndoG also showed changes in cell distribution in the cell cycle, producing a pattern characteristic of cells that have been arrested in the G(2) phase. These findings suggest that EndoG is required for normal cellular proliferation.

Fig. 1.

Human EndoG nucleotide sequences and the three 19-mers selected as shRNA targets. Nucleotide sequences (underlined) at positions 28–46 (T1), 682–700 (T3), and 693–711 with an A-to-T mutation at 708 (T5m) were selected to construct pT3, pT1, and pT5m with pS as the plasmid backbone. The translation start site (bold ATG) is at 167–169. T3 and T5m overlap by 8 nucleotides (dashed line). These sequences were also used to construct pT1-RT, pT3-RT, and pT5m-RT, with pS-RT as the plasmid backbone.

Fig. 2.

Reduction of EndoG by RNA interference. (A) Agarose gel electrophoresis showing RT-PCR products obtained from cells transfected for 5 days with pS, pT1, pT3, and pT5m in the presence of G418. The positions of the 241-bp fragment representing EndoG and the 465-bp fragment representing the internal control human porphobilinogen deaminase (hPBGD) are indicated. (B) Intensity of the EndoG band of each sample, measured and normalized to that of the hPBGD band. The normalized EndoG value of each sample was compared with that of the cells containing pS.

Fig. 3.

Analysis of a sequence-mediated recombination. (A) Illustration of the recombination reaction. (a) A pRD105 concatemer showing two units of pRD105 (P). Dark arrows represent HSV-1 a sequences in a direct-repeat orientation. The locations of the a sequences are indicated as 1–5. NheI cutting sites, a unique site on each pRD105, are indicated as vertical lines. Predicted products (R1 and R2) resulting from recombination reactions between a sequence pairs in locations 1 and 2, 2 and 3, and 1 and 3 are indicated in b, c, and d respectively. (B) Autoradiogram showing the positions of pRD105 (P) and the recombination products R1 (11.3 kb) and R2 (3 kb). Vero cells were transfected with the indicated plasmids for 5 days and then infected with a mixture of HSV-1 and HSV-1 defective particles containing pRD105 concatemers. After infection for 6 and 12 h, the DNA was extracted and analyzed for pRD105 (P) and the recombination product R2 by Southern blotting followed by hybridization to a ³²P-labeled probe, which hybridized to a region in the 3-kb (R2) fragment. (C) Summary of recombination products from five experiments as described in B. Recombination efficiency is indicated as the R2:P ratio. (D) Statistical analysis (t test) of differences in recombination efficiency.

Fig. 4.

Effect of reduction of EndoG on cellular proliferation. Growth of cells transfected with pS, pT1, pT3, and pT5m (Fig. 2) was measured in medium containing G418. The -fold increase was determined by comparing the live-cell number when RT-PCR was performed (day 5) with the number of cells seeded 1 day after transfection (day 1). Untransfected cells were undetectable.

Fig. 5.

Rescue of cell-proliferation defect by exogenous expression of EndoG-GFP. (A) Summary of live-cell (293T) number relative to that of control cells cotransfected with pS-RT/pRK5 (bar 1), an empty vector that shares a promoter structure similar to that of pEndoG-GFP. Each bar represents cells transfected with 400 ng of each plasmid. Bar 2, cells cotransfected with pS-RT/pEndoG-GFP. Bars 3 and 4, cells cotransfected with pT5m-RT/pRK5 and pT5m-RT/pEndoG-GFP. (B) Summary of live-cell (293T) number relative to the control (bar 1). Experiments were performed as described in A except 800 ng of each plasmid was used for transfection.

Fig. 6.

Distribution of 293T cells transfected with pS-RT and pT5m-RT during the cell cycle. (A) FACS histograms show that pS-RT- and pT5m-RT-transfected cells (stained with PI) distribute differently in the cell cycle. (B) Summary of cell numbers distributed in the G₀/G₁, S, and G₂/M phases of the cell cycle from three individual experiments as described in A.