Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila

Abstract

DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or “alternative” end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s) are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta), encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair.

Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids

“…Polθ has multiple documented activities in DNA replication and repair, including alt-EJ, replication repair, translesion synthesis, and replication initiation (4)(5)(6)(7)(8)10,15,31,32). Although the activities and cellular functions of Polθ-polymerase have been investigated, little is understood regarding the enzymatic activities of Polθ-helicase.…”

“…1a) (1)(2)(3). Understanding the biochemical activities and cellular functions of Polθ have become a priority because it has been found to be essential for the error-prone doublestrand break (DSB) repair pathway known as microhomology-mediated end-joining (MMEJ) or alternative end-joining (alt-EJ) (3)(4)(5)(6)(7)(8)(9). Remarkably, Polθ expression has also been shown to be important for the proliferation of cells deficient in the homologous recombination (HR) pathway, such as due to mutations in BRCA1 or BRCA2 (4,10).…”

Essential role for polymerase specialization in cellular nonhomologous end joining

“…Strikingly, one other eukaryotic polymerase has a related activity-Pol θ also directs synthesis across double-strand break ends, although it needs a short patch of terminal complementary sequence, or microhomology, to initiate synthesis-and it is essential for an alternate NHEJ pathway (29)(30)(31). More complex and longer-lived organisms are expected to need more flexible and efficient versions of end joining; it is clear that the use of specialized polymerases is an integral part of how this has been achieved.…”

A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites

“…Mammalian Pol y has the ability to bypass DNA lesions and to extend matched as well as mismatched primer termini 26,27 . In addition, Drosophila Pol y was recently implicated in the repair of DNA DSBs induced by DNA transposition 28 . To test a possible involvement of the C. elegans homologue of Pol y, POLQ-1, we first used a PCR-based assay that detects G4 DNA-induced deletions in isolated DNA by preferential amplification of smaller than wild-type bands of sequences containing G4 motifs 17 .…”