Mammalian polymerase θ promotes alternative nhej and suppresses recombination

ABSTRACT The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair

pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions1,2,3. Using next-generation sequencing technology, here we show that

repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random

insertions enabled us to identify polymerase theta (Polθ; encoded by _Polq_ in mice) as a crucial alternative NHEJ factor in mammalian cells. _Polq_ inhibition suppresses alternative NHEJ at

dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of _Polq_ in mice results in increased rates of homology-directed

repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell

survival in the absence of _BRCA_ genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed

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Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS REPAIR OF G1 INDUCED DNA DOUBLE-STRAND BREAKS IN S-G2/M BY ALTERNATIVE NHEJ Article Open access 16 October 2020 PATHWAY CHOICE

IN THE ALTERNATIVE TELOMERE LENGTHENING IN NEOPLASIA IS DICTATED BY REPLICATION FORK PROCESSING MEDIATED BY EXD2’S NUCLEASE ACTIVITY Article Open access 27 April 2023 POLΘ: EMERGING

SYNTHETIC LETHAL PARTNER IN HOMOLOGOUS RECOMBINATION-DEFICIENT TUMORS Article Open access 09 August 2024 ACCESSION CODES PRIMARY ACCESSIONS BIOPROJECT * PRJNA269507 DATA DEPOSITS Sequence

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1657–1661 (2010) Article  ADS  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank T. de Lange, R. Greenberg, J. Shay, N. Shima, C. Cazaux and R. Wood for providing key

reagents for this study. We are grateful to M. Ji, L. Walton Masters, A. Phillips, A. Pinzaru, F. Yeung, P. Tonzi and J. Wong for technical assistance. We thank S. Kabir and F. Lottersberger

for critical reading of the manuscript. This work was supported by a grant from the Breast Cancer Alliance (A.S.), V-foundation (A.S.), Department of Defense Breast Cancer Research Program

BC134020 (P.A.M.-G.), Pew-Stewart Scholars Award (A.S.), Pew Scholars Award (E.L.-D.), Novartis Advanced Discovery Institute (E.L.-D.), and a grant from the National Institutes of Health

(NIH) AG038677 (E.L.-D.). The A.S. laboratory was supported by start-up funds from the Helen L. and Martin S. Kimmel Center for Stem Cell Biology. The K.M.M. laboratory was supported in part

by start-up funds from the University of Texas at Austin and from the Cancer Prevention Research Institute of Texas (CPRIT, R116). K.M.M. is a CPRIT scholar. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Cell Biology, Skirball Institute of Biomolecular Medicine, NYU School of Medicine, New York, 10016, New York, USA Pedro A. Mateos-Gomez & Agnel Sfeir *

Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin. 2506 Speedway Stop A5000, Austin, 78712, Texas, USA Fade Gong & Kyle M.

Miller * Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, 92037, California, USA Nidhi Nair & Eros Lazzerini-Denchi Authors * Pedro A.

Mateos-Gomez View author publications You can also search for this author inPubMed Google Scholar * Fade Gong View author publications You can also search for this author inPubMed Google

Scholar * Nidhi Nair View author publications You can also search for this author inPubMed Google Scholar * Kyle M. Miller View author publications You can also search for this author

inPubMed Google Scholar * Eros Lazzerini-Denchi View author publications You can also search for this author inPubMed Google Scholar * Agnel Sfeir View author publications You can also

search for this author inPubMed Google Scholar CONTRIBUTIONS A.S., E.L.-D. and P.A.M.-G. conceived the experimental design. P.A.M.-G. and A.S. performed the experiments and analysed the

data. E.L.-D. and N.N. performed telomere-sequencing experiments. F.G. and K.M.M. performed experiments related to Polθ localization at DNA breaks. A.S. wrote the manuscript. All authors

discussed the results and commented on the manuscript. CORRESPONDING AUTHOR Correspondence to Agnel Sfeir. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial

interests. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 POLΘ PROMOTES ALT-NHEJ REPAIR AT DYSFUNCTIONAL TELOMERES. (Related to Fig. 1.) A, Immunoblots for TRF1 and RAP1 after

4-OHT-induced depletion of TRF2 from _Trf2__F/F_Cre-ERT2 MEFs and co-depletion of TRF1 and TRF2 from _Trf1__F/F __Trf2__F/F __Ku80__−/−_ Cre-ERT2 cells. Loss of TRF2 is confirmed by the

disappearance of RAP1; a TRF2-interacting protein the stability of which depends on TRF2 (refs 35, 36). B, To validate the effect of _Polq_ depletion on alt-NHEJ we monitored the frequency

of telomere fusions in shelterin-free _Ku80_-null cells treated with three independent shPolq vectors. shPolq-1 was used in Fig. 2. Mean values are presented with error bars denoting ± 

s.e.m. from two independent experiments. Source data EXTENDED DATA FIGURE 2 POLΘ DRIVES CHROMOSOMAL TRANSLOCATIONS IN MOUSE CELLS. (Related to Fig. 2.) A, Immunobloting for Polθ in MEFs with

the indicated genotype and treatment. B, Immunoblot for TRF1 in MEFs with the indicated genotype. Cells were analysed 96 h after Cre induction. C, RAP1 immunoblot (similar to B). D, Western

blot analysis for Polθ and Flag–Cas9 in lysates prepared from _Polq__−/−_ and _Polq_+/+ cells after Cas9 expression. Tubulin serves as a loading control. E, Surveyor nuclease assay for

_Polq__−/−_ and _Polq_+/+ cells expressing Cas9-gRNA(Rosa26;H3f3b) plasmid. Genomic DNA isolated from cells with the indicated genotype was used as a template to amplify across the cleavage

site at either the Rosa26 or the H3f3b locus to assess intra-chromosomal NHEJ. Amplification products were denatured and then re-annealed to form heteroduplexes between unmodified and

modified sequences from imprecise NHEJ. The mismatched duplex was selectively cleaved by the Surveyor nuclease at the loops that form at mismatches. F, Signature of translocations in

_Polq__−/−_ and _Polq_+/+ cells (see Extended Data Figs 3–4, 5 for complete list of sequences). Table records the total number of translocation events identified following CRISPR-Cas9

induced-cleavage. On average, the same number of nucleotides was deleted at the fusion junction in _Polq__−/−_ and _Polq_+/+ cells. No nucleotide insertions were found in the absence of

_Polq_. Lastly, the percentage of junctions exhibiting microhomology was significantly reduced in cells lacking _Polq_. G, Scheme depicting Polθ domains. CRISPR/Cas9 gene targeting was used

to create two base substitutions at Asp2494Gly and Glu2495Ser, and generate a catalytic-dead polymerase34. H, Sequence analysis of targeted cells. I, Genotyping PCRs of _Polq_+/+ and

_Polq__CD_ (catalytically dead allele of _Polq_) after SacII digestion. J, Immunoblotting to analyse Cas9 expression in _Polq_+/+ and two independently derived _Polq__CD_ clonal cell lines.

K, Frequency of chromosomal translocations (der(6)) in _Polq_+/+ and _Polq__CD_ cells. Bars represent mean of four independent experiments ± s.d. (two experiments per clonal cell line). _P_

= 0.006; two-tailed Student’s _t_-test. PCR products were sequenced to confirm translocation and identify possible insertions. Source data EXTENDED DATA FIGURE 3 SEQUENCE ANALYSIS OF

TRANSLOCATION JUNCTIONS IN _POLQ_+/+ CELLS. (Related to Fig. 2.) Sequences of der(11) breakpoint junction from _Polq_+/+ cells. Predicted fusion breakpoint based on CRISPR cutting indicated

by an arrow. Reference sequence highlighted at the top. The remaining lines represent individual translocations recovered by PCR and subject to Sanger sequencing. Nucleotide insertions are

marked in red. In cases where insertions extended beyond the sequence included in the lane, the length of the insertion was noted in parenthesis (red). Gaps in the sequence represent

nucleotide deletions. The average length of the deletions was noted in Extended Data Fig. 2f. Micro-homology is denoted by blue boxes. Micro-homology embedded in DNA extending beyond the

enclosed sequence was noted in parentheses (blue). EXTENDED DATA FIGURE 4 SEQUENCE ANALYSIS OF TRANSLOCATION JUNCTIONS IN _POLQ_+/+ CELLS. (Related to Fig. 2.) Sequences of der(6) breakpoint

junction from _Polq_+/+ cells. Predicted fusion breakpoint based on CRISPR cutting indicated by an arrow. Reference sequence highlighted at the top. The remaining lines represent individual

translocations recovered by PCR and subject to Sanger sequencing. EXTENDED DATA FIGURE 5 SEQUENCE ANALYSIS OF TRANSLOCATION JUNCTIONS IN _POLQ__−/−_ CELLS. (Related to Fig. 2.) Sequences of

der(11) and der(6) breakpoint junction from _Polq__−/−_ cells. Predicted fusion breakpoint based on CRISPR cutting indicated by an arrow. Reference sequence is highlighted at the top. The

remaining lines represent individual translocations recovered by PCR and subject to Sanger sequencing. It is important to note that insertions were completely lacking at the fusions

junctions in _Polq__−/−_ cells. EXTENDED DATA FIGURE 6 POLΘ RECRUITMENT TO DNA BREAKS. (Related to Fig. 3.) A, Laser micro-irradiation experiment using HeLa cells expressing Myc–Polθ and

treated with ATM inhibitor (KU55933), ATR inhibitor (VE-821) or PARP inhibitor (KU58948). B, Western blot analysis for CHK1 and CHK2 phosphorylation. Cells with the indicated treatment were

analysed 2 h after irradiation. C, Immunoblot for PARP1. HeLa cells were treated with PARP1 siRNA and analysed 72 h after siRNA transfection for efficiency of knockdown. Source data EXTENDED

DATA FIGURE 7 PARP1-DEPENDENT POLΘ RECRUITMENT TO DNA DOUBLE-STRANDED BREAKS (DSBS). (Related to Fig. 3.) A, Results from immunofluorescence performed 4 h after induction (1 µM Shield1

ligand, Clontech 631037; 0.5 μM 4-OH tamoxifen) of DSBs by mCherry-LacI-FokI in the U2OS-DSB reporter cells18 transfected with the Myc–Polθ and treated with PARP inhibitor (KU58948). The

mCherry signal is used to identify the area of damage and to assess the recruitment of Myc–Polθ to cleaved LacO repeats. B, Table displaying quantification related to A. Source data EXTENDED

DATA FIGURE 8 POLΘ SUPPRESSES HOMOLOGY-DIRECTED REPAIR AT DYSFUNCTIONAL TELOMERES. (Related to Fig. 3.) A, Western blot analysis for Polθ and LIG3 in shelterin-free _Lig4_-null MEFs. B,

Western blot for TRF1 and RAP1 after 4-OHT treatment of shelterin-free _Lig4_-deficient cells. C, Metaphase spreads from _Trf1__F/F __Trf2__F/F __Lig4__−/−_ Cre-ERT2 MEFs, with the indicated

shRNA treatment, 96 h after Cre expression. CO-FISH assay was performed using a FITC-OO-(CCCTAA)3 PNA probe (green) and a Tamra-OO-(TTAGGG)3 PNA probe (red). DAPI in blue. Examples of

alt-NHEJ-mediated fusion and T-SCE events (HDR) are indicated by white and red arrows, respectively. Examples of T-SCE events reflective of increased HDR in cells treated with shPolq are on

the right. D, E, Quantification of telomere fusions by alt-NHEJ in MEFs with the indicated genotype and shRNA treatment. Bars represent mean of two independent experiments ± s.e.m. F,

Representative in-gel hybridization to assess 3′ overhang of _Trf1__F/F __Trf2__F/F __Lig4__−/−_ Cre-ERT2 MEFs with the indicated shRNA treatment after Cre deletion. G, Quantification of the

gel in F. The single-stranded DNA/total signal ratios of the ‘+Cre’ samples are expressed relative to the ‘−Cre’ samples for each shRNA treatment. Mean of two independent experiments. H,

Graph representing RAD51 accumulation after ionizing radiation treatment of _Polq__CD_, _Polq_+/+ and _Polq__−/−_ embryonic stem cells. Bars represent mean of two independent experiments.

_P_ >0.05; two-tailed Student’s _t_-test. Source data EXTENDED DATA FIGURE 9 POLΘ PROMOTES ALT-NHEJ AND INHIBITS HOMOLOGY-DIRECTED REPAIR AT I-SCEI-INDUCED DNA BREAKS. (Related to Fig.

3.) A, Polθ represses recombination at DSBs induced by I-Sce1. The TLR system was used to measure the relative ratio of end-joining (mCherry) and HDR (enhanced green fluorescent protein

(eGFP)) repair of a DSB. A diagram of the TLR is represented. B, The TLR construct was stably integrated into _Lig4__−/_ and _Ku80__−/−_ MEFs to avoid the confounding effect of C-NHEJ, and

limit end-joining reactions to the alt-NHEJ pathway. Expression of mCherry and eGFP was assessed by flow cytometry 72 h after I-Sce1 and 5′ eGFP donor transduction in cells with the

indicated shRNA construct. Percentages of cells are indicated in the plot. C, Quantification of alt-NHEJ and HDR of TLR containing _Ku80__−/−_MEFs after expression of I-Sce1 and 5′ eGFP

together with the indicated shRNA construct. Bar graphs represent the mean of three independent experiments ± s.d. _P_ = 0.03; two-tailed Student’s _t_-test. D, Real-time PCR to monitor the

knockdown efficiency of _Polq_ in _Ku80__−/−_ and _Lig4__−/−_ MEFs. The FACS analysis reported in E and F was carried out without selecting for cells expressing the shRNA-containing plasmid.

Source data EXTENDED DATA FIGURE 10 POLΘ IS REQUIRED FOR SURVIVAL OF RECOMBINATION-DEFICIENT CELLS. (Related to Fig. 4.) A, Accumulation of chromosomal aberrancies after _Brca1_ and _Brca2_

knockdown in _Polq__−/−_ and _Polq_+/+ MEFs. Quantification of chromosomal aberrancies (chromatid breaks, chromosome breaks and radials) in MEFs stably transduced with lentiviral vectors

expressing the indicated shRNA. B, Real-time PCR to confirm the knockdown of _Brca1_ and _Brca2_ as in A. C, Quantitative analysis of colony formation in _Brca1__F/F_ Cre-ERT2 and

_Lig4__−/−_ cells after _Polq_ depletion. The number of colonies in control shRNA-treated cells was set to 100%. Mean values are presented with error bars denoting ± s.d. from three

independent experiments. D, Real-time PCR to measure the knockdown efficiency of human _POLQ_ in BJ-hTERT, MCF7 and HCC1937 cells and mouse _Polq_ in _Brca1__F/F_ Cre-ERT2 cells. E,

Quantitative analyses of colony formation in BJ-hTERT, MCF7 and HCC1937 cells after LIG3 inhibition. The number of colonies in control-shRNA-treated cells was set to 100%. The knockdown

efficiency for _Lig3_ was ∼85%. Bars represent mean of two independent experiments ± s.e.m. F. Quantitative analyses of colony formation in _Polq__CD_ and _Polq_+/+ embryonic stem cells

after _BRCA1_ inhibition. The number of colonies in control-shRNA-treated cells was set to 100%. The knockdown efficiency for _BRCA1_ was >80%. Bars represent mean of two independent

experiments ± s.e.m. _P_ = 0.05; two-tailed Student’s _t_-test. Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION This file contains Supplementary Data including sequence

analysis of telomere fusions using illumina technology and C‐NHEJ junction sequences. (PDF 233 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE

FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 SOURCE DATA SOURCE DATA TO FIG. 1 SOURCE DATA TO FIG. 2 SOURCE DATA TO FIG. 3 SOURCE DATA TO FIG. 4 SOURCE DATA TO EXTENDED DATA FIG. 5 SOURCE DATA TO

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DATA FIG. 11 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Mateos-Gomez, P., Gong, F., Nair, N. _et al._ Mammalian polymerase θ promotes alternative

NHEJ and suppresses recombination. _Nature_ 518, 254–257 (2015). https://doi.org/10.1038/nature14157 Download citation * Received: 28 July 2014 * Accepted: 16 December 2014 * Published: 02

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