Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination

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 fusions. 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 repair genes.

[1]  M. Tijsterman,et al.  Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans , 2014, Genome research.

[2]  T. Kunkel,et al.  Low-fidelity DNA synthesis by human DNA polymerase theta , 2008, Nucleic acids research.

[3]  A. Multani,et al.  The function of classical and alternative non‐homologous end‐joining pathways in the fusion of dysfunctional telomeres , 2010, The EMBO journal.

[4]  M. Hatano,et al.  DNA polymerase θ is preferentially expressed in lymphoid tissues and upregulated in human cancers , 2004, International journal of cancer.

[5]  S. Powell,et al.  Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells , 2013, Nucleic acids research.

[6]  E. Schwob,et al.  A role for DNA polymerase θ in the timing of DNA replication , 2014, Nature Communications.

[7]  T. de Lange,et al.  Removal of Shelterin Reveals the Telomere End-Protection Problem , 2012, Science.

[8]  David J. Rawlings,et al.  Tracking genome engineering outcome at individual DNA breakpoints , 2011, Nature Methods.

[9]  J. Diedrich,et al.  A two-step mechanism for TRF2-mediated chromosome end protection , 2013, Nature.

[10]  L. Symington,et al.  RPA coordinates DNA end resection and prevents formation of DNA hairpins. , 2013, Molecular cell.

[11]  M. Haughton,et al.  The nature of telomere fusion and a definition of the critical telomere length in human cells. , 2007, Genes & development.

[12]  Michael M. Murphy,et al.  IgH class switching and translocations use a robust non-classical end-joining pathway , 2007, Nature.

[13]  George Iliakis,et al.  PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways , 2006, Nucleic acids research.

[14]  Wouter Koole,et al.  A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites , 2014, Nature Communications.

[15]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[16]  T. Lange,et al.  DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion , 2005, Nature Cell Biology.

[17]  J. A. Halliday,et al.  Engineered proteins detect spontaneous DNA breakage in human and bacterial cells , 2013, eLife.

[18]  R. Greenberg,et al.  Acetylation Limits 53BP1 Association with Damaged Chromatin to Promote Homologous Recombination , 2012, Nature Structural &Molecular Biology.

[19]  Bas van Steensel,et al.  TRF2 Protects Human Telomeres from End-to-End Fusions , 1998, Cell.

[20]  M. McVey,et al.  Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila , 2010, PLoS genetics.

[21]  M. Weinfeld,et al.  Involvement of polynucleotide kinase in a poly(ADP-ribose) polymerase-1-dependent DNA double-strand breaks rejoining pathway. , 2006, Journal of molecular biology.

[22]  S M Bailey,et al.  Strand-Specific Postreplicative Processing of Mammalian Telomeres , 2001, Science.

[23]  Huichen Wang,et al.  DNA ligase III as a candidate component of backup pathways of nonhomologous end joining. , 2005, Cancer research.

[24]  E. Johansson,et al.  Promiscuous DNA synthesis by human DNA polymerase θ , 2011, Nucleic acids research.

[25]  Eric C Greene,et al.  RPA Antagonizes Microhomology-Mediated Repair of DNA Double-Strand Breaks , 2014, Nature Structural &Molecular Biology.

[26]  Samuel H. Wilson,et al.  Human DNA polymerase θ possesses 5′-dRP lyase activity and functions in single-nucleotide base excision repair in vitro , 2009, Nucleic acids research.

[27]  J. Schimenti,et al.  Phenotype-based identification of mouse chromosome instability mutants. , 2003, Genetics.

[28]  A. Thompson,et al.  DNA polymerase θ up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability , 2010, Proceedings of the National Academy of Sciences.

[29]  G. Celli,et al.  Loss of Rap1 Induces Telomere Recombination in the Absence of NHEJ or a DNA Damage Signal , 2010, Science.

[30]  A. Sfeir,et al.  Telomeres at a glance , 2012, Journal of Cell Science.

[31]  Lei Zhang,et al.  DNA Ligase III Promotes Alternative Nonhomologous End-Joining during Chromosomal Translocation Formation , 2011, PLoS genetics.

[32]  Linda Z. Shi,et al.  Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells , 2013, Proceedings of the National Academy of Sciences.

[33]  M. Jasin,et al.  Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4/ligase IV during chromosomal translocation formation , 2010, Nature Structural &Molecular Biology.

[34]  Ilya J. Finkelstein,et al.  Nucleosome Acidic Patch Promotes RNF168- and RING1B/BMI1-Dependent H2AX and H2A Ubiquitination and DNA Damage Signaling , 2014, PLoS genetics.

[35]  Thomas Helleday,et al.  Overexpression of POLQ Confers a Poor Prognosis in Early Breast Cancer Patients , 2010, Oncotarget.

[36]  F. Couch,et al.  Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers , 2008, Nature.