Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction.
Here researchers at the University of North Carolina at Chapel Hill used OligoMix as a sgGuide library to develop a CRISPR genetic screen that uncovered 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicated that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. The researchers further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.
a Schematic of the CRISPR genetic screen to identify PolqSL genes. b Violin plot of Gene Abundance Change Scores (Log2) for DDR gene-targeting sgRNAs (red) and non-targeting control sgRNAs (blue) in Polq−/− and PolqhPOLQ MEFs, relative to WT MEFs. c Volcano plot of Gene Abundance Change Scores (Polq−/− versus PolqhPOLQ) and -Log10 p-value of the Kolmogorov-Smirnov test for DDR gene-targeting sgRNAs relative to non-targeting control sgRNAs. Thresholds for statistical significance are indicated by dashed lines (see “Methods” for details). Genes with statistically significant (Blue dots) and non-significant (Gray dots) Gene Abundance Changes Scores are indicated. d Relative cell survival measured by colony forming efficiency of WT or Polq−/− cells transduced with a lentivirus containing Cas9 and control sgRNA (sgCtrl) or DDR gene-targeting sgRNAs. Data shown are the mean ± SEM (n = 3 biological replicates). Significance determined using an unpaired, two-tailed t-test (*p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001). e Functional classification of PolqSL genes identified in our CRISPR screen depicted as a Euler diagram
Feng W, Simpson DA, Carvajal-Garcia J, Price BA, Kumar RJ, Mose LE, Wood RD, Rashid N, Purvis JE, Parker JS, Ramsden DA, Gupta GP. (2019) Genetic determinants of cellular addiction to DNA polymerase theta. Nat Commun 10(1):4286. [article]