Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

Abstract

DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome.

Figure 1. Partial characterization of DSB repair complexes by co-IP analysis.

NEs (benzonase-treated) from HEK293 cells (either mock (−) or Bleo- (+) treated, or allowed to recover (+/R, 10–12 h) after Bleo treatment) were immunoprecipitated (IP'd) with (a) anti-RNAP II (pSer2, H5 Ab); (b) anti-53BP1; (c) anti-Lig IV; (d) anti-PNKP; (e) anti-PARP1; or (f) anti-Lig IIIα antibodies (Abs, even lanes) or control IgG (odd lanes) and tested for the presence of associated proteins with specific Abs as indicated to the right of each row. Individual IP experiments were repeated at least three times from separate batches of cells, and one representative figure is shown in each case.

Figure 2. Quantitative ChIP assay to determine the association of C-NHEJ proteins with transcribed versus non-transcribed genes.

(a) HEK293 cells were mock-, GO- or Bleo-treated after mock (−) or DRB (+) treatment. ChIP was performed with specific Abs as indicated, and binding to the exonic regions of transcribed (GAPDH and ß-Actin) versus non-transcribed (NANOG and NeuroD) genes was quantified by qPCR from IP'd DNA. The data are represented as fold enrichment of per cent input over IgG. Error bars represent±s.d. of the mean (n≥3). ***P<0.005 represents statistical significance within a treatment group between a particular transcribed gene and both non-transcribed genes (NANOG and NeuroD) (b) DNA DSBs were generated at sites A (transcribing) and B (non-transcribing) on chromosome number 1 of human U2OS cells and ChIP was performed with Lig IV/Lig IIIα before and after CRISPR/Cas9-induction of DSBs. The data represent the relative levels of association of Lig IIIα/IV (mean±s.d.) on the DSB sites. n=3,*P<0.05; **P<0.01; ***P<0.005. (c) HEK293 cells were either mock-treated (-HS) or subjected to heat shock (+HS), or DRB treatment followed by HS (DRB+HS). The cells were further mock/Bleo-treated and ChIP was performed with specific Abs or control IgG. Binding to the HSP70 and HSP89 gene(s) was quantified by qPCR from IP'd DNA. Per cent input over IgG was calculated and represented as fold increase with -HS samples as unity. Error bars represent±s.d. of the mean (n≥3). The data are significant at ***P<0.005 between each set of –HS/+HS and +HS/DRB+HS. (d) Long amplicon quantitative PCR-mediated estimation of DNA damage in the HSP70 gene before (-HS) and after heat-shock (+HS) treatment. (–) and (+) represent mock and Bleo treatment, respectively. In each case, the short genomic fragment (∼200 bp) is amplified (SA-PCR) for normalization of the LA-qPCR data; the bar diagram represents normalized band intensity (mean±s.d.; n=3).

Figure 3. Evaluation of genomic strand-break levels in transcribed versus non-transcribed genes by LA-qPCR.

HEK293 cells were transfected with either control siRNA or that specific for 53BP1 (a), Lig IV (b) or PNKP (c) and further mock- (−) or Bleo- (+) treated or kept for recovery (R) after Bleo treatment, and harvested for genomic DNA isolation. In another case, HEK293 cells ectopically expressing PNKP were transfected with either control siRNA or that specific for the PNKP 3′UTR (d) for depletion of endogenous PNKP, followed by mock/Bleo treatment as above. Amplification of each large fragment (8–12 kb) was normalized to that of a small fragment (∼200–400 bp) of the corresponding transcribed (HPRT1 and POLB) or non-transcribed (NANOG and OCT3/4) gene; the normalized data are represented (in the bar diagram) as relative band intensity with the control siRNA/mock-treated sample arbitrarily set as 100 (n≥3; one representative gel figure is shown). Error bars represent±s.d. of the mean. The persistence of damage after recovery for each transcribed gene (panels a, b and c; HPRT1 and POLB) is significant (***P<0.005) compared with corresponding mock-treated samples; however, the damage is almost completely repaired in non-transcribed genes (NS, non-significant, P>0.05; *P<0.05).

Figure 4. C-NHEJ pathway utilizes RNA as a template for error-free repair.

(a) HEK293 cells were mock-treated or treated with Bleo or GO and their NEs subjected to RNA-ChIP analysis using the indicated Abs. RT-PCR was carried out for TUBB, HPRT1, POLB, USP5 and HSP90B genes using intron-specific primers. Lanes: 1, 7 and 13 represent 1% inputs, collected before IPs, and M shows a 1 kb DNA ladder. The image shown here is representative of plus reverse transcriptase (+RT) reactions in three independent experiments. (b) Time-course analysis, where 1 h Bleo-treated HEK293 cells were washed with PBS before they were allowed to recover for the indicated times, and NEs were subjected to RNA-ChIP using anti-PNKP or -53BP1 Abs. Real-time assays were done using intron-specific primers for TUBB and HPRT1, and per cent inputs were calculated using C_t values; error bars represent ±s.d. of the mean (n≥3). The dissociation of pre-mRNA from the complex at 3 and 6 h is significant (**P<0.01; ***P<0.005) compared with 0 h. (c) RNA-ChIP analysis was done with NE from mock-, Bleo- or GO-treated HEK293 cells using anti-RAD51 and -RAD52 Abs, then RT-PCRs were performed with intron-specific primers for the indicated genes. Lanes 1, 5 and 9 shows 1% input collected before IPs. Representative images for +RT reactions in three independent experiments are shown here. (d) Bleo-treated cells were incubated with RNase H, before RNA-ChIP assays using Abs against PNKP and 53BP1, to detect RNA–DNA hybrids and real-time qPCRs were done using intron-specific primers for TUBB or HPRT1. Data are presented as per cent input where error bars show ±s.d. of the mean (n=3, **P<0.01, ***P<0.005).

Figure 5. RNA-templated in vitro and plasmid-based in cell DSBR assays.

Twenty picomol of RNA-templated substrate (shown schematically in a) were used to assess total repair activity (b) in the NEs from control and PNKP siRNA-transfected HEK293 cells (lanes 3 and 4). Similar assays were performed with NEs from control or PNKP siRNA-transfected HEK293 cells in DNA-templated (lanes 1 and 2; as control) substrates (5 pmol), and an RNase A-treated RNA-templated substrate in control HEK293 cells (lane 7). A similar total repair assay was also carried out in mock (−)/Line1 specific RT inhibitor-treated (+) NEs with RNA-templated substrates (lanes 5 and 6). The upper arrow indicates the 51-mer repaired product, and the lower arrow, un-ligated product (n=3 in each case, and one representative gel is shown). 5′ end-labelled 25 nt (lane 8) and 51 nt (lane 9) oligos were used as markers. The repair efficiency (in terms of per cent ligated product) was not significantly different between mock and RT inhibitor (RTI)-treated NE (NS, non-significant, P>0.05). (c) Plasmid-based DSBR assay in mammalian cells, measured by the number of colonies carrying the repaired plasmid in E. coli. The data are represented as the fold increase in the number of E. coli blue colonies harbouring plasmid DNA that was recovered after 16 h of repair in HEK293-TetON^+DoxlacZ versus HEK293-TetON^−DoxlacZ stable cell lines (after normalizing the transfection efficiency). The number of E. coli blue colonies after transformation of the plasmids from HEK293-TetON^−DoxlacZ was arbitrarily set as 1. The data are the average of at least 3 independent experiments (upper panel) where ***P<0.005. The lower panel depicts RT-PCR showing the expression of lacZ in HEK293 ±Doxycycline (Dox) TetONlacZ cells. M, 2-log DNA Ladder from New England Biolabs. (d) Plasmid-based DSBR assay was performed as in c, but in stably expressing HEK293-Pcmv⁺lacZ cells treated with the indicated siRNA. The number of E. coli blue colonies after transformation of the plasmids from cells treated with control siRNA was arbitrarily set as 1. The data are the average of at least three independent experiments; ***P<0.005 and NS, non-significant (P>0.05). The comparison was done in each case between control and specific siRNA-treated samples.

Figure 6. Model of nascent RNA-mediated C-NHEJ.

A model depicting the role of pre-mRNA in providing the template for C-NHEJ-mediated error-free repair.