Abstract
Homologous recombination-mediated gene targeting has greatly contributed to genetic analysis in a wide range of species, but is highly inefficient in human cells because of overwhelmingly frequent random integration events, whose molecular mechanism remains elusive. Here we show that DNA polymerase θ, despite its minor role in chromosomal DNA repair, substantially contributes to random integration, and that cells lacking both DNA polymerase θ and DNA ligase IV, which is essential for non-homologous end joining (NHEJ), exhibit 100% efficiency of spontaneous gene targeting by virtue of undetectable levels of random integration. Thus, DNA polymerase θ-mediated end joining is the sole homology-independent repair route in the absence of NHEJ and, intriguingly, their combined absence reveals rare Alu-Alu recombination events utilizing a stretch of homology. Our findings provide new insights into the mechanics of foreign DNA integration and the role of DNA polymerase θ in human genome maintenance.
Highlights
Homologous recombination-mediated gene targeting has greatly contributed to genetic analysis in a wide range of species, but is highly inefficient in human cells because of overwhelmingly frequent random integration events, whose molecular mechanism remains elusive
To investigate the mechanism of Lig4-independent random integration (RI), we used a promoterless targeting vector, p4.5 HPRT-2A-EGFP-2A-Puro[35], to perform gene targeting in LIG4-knockout cells derived from a human diploid cell line, Nalm-6, and sought to characterize the nature of random integrants (‘RI clones’) arising in an non-homologous end joining (NHEJ)-independent manner (Fig. 1a and Supplementary Fig. 1)
Loss of polymerase y (Pol y) is synthetically lethal with homologous recombination (HR) defects[33,47] and developing Pol y inhibitors will lead to a new therapeutic agent for various cancers with compromised HR40,48
Summary
Homologous recombination-mediated gene targeting has greatly contributed to genetic analysis in a wide range of species, but is highly inefficient in human cells because of overwhelmingly frequent random integration events, whose molecular mechanism remains elusive. We show that DNA polymerase y, despite its minor role in chromosomal DNA repair, substantially contributes to random integration, and that cells lacking both DNA polymerase y and DNA ligase IV, which is essential for non-homologous end joining (NHEJ), exhibit 100% efficiency of spontaneous gene targeting by virtue of undetectable levels of random integration. Despite its minor role in chromosomal DSB repair, Pol y substantially contributes to RI and cells doubly deficient in Pol y and Lig[4] exhibit 100% gene-targeting efficiency because of virtually no RI events. Our findings provide new insights into the mechanics of foreign DNA integration and the role of Pol y in preserving genome integrity
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