DNA polymerase theta (Polθ) - an error-prone polymerase necessary for genome stability

Abstract

Mammalian cells have evolved multiple pathways to repair DNA double strand breaks (DSBs) and ensure genome stability. In addition to non-homologous end-joining (NHEJ) and homologous recombination (HR), cells evolved an error-prone repair pathway termed microhomology-mediated end joining (MMEJ). The mutagenic outcome of MMEJ derives from the activity of DNA polymerase theta (Polθ) - a multidomain enzyme that is minimally expressed in normal tissue but overexpressed in tumors. Polθ expression is particularly crucial for the proliferation of HR deficient cancer cells. As a result, this mutagenic repair emerged as an attractive target for cancer therapy, and inhibitors are currently in pre-clinical development. Here, we review the multifunctionality of this enigmatic polymerase, focusing on its role during DSB repair in mammalian cells and its impact on cancer genomes.

Figure 1.. Major DSB repair pathways in mammalian cells.

DSBs in G1 are primarily repaired by NHEJ. The Ku heterodimer recognizes broken DNA and recruits DNA-PKcs to orchestrate end-joining by Lig4. During NHEJ, minimal processing of DNA ends leads to repair with minimal alteration to the original sequence. DSBs in S/G2 phases of the cell cycle are subjected to end-resection by MRN/CtIP, leading to short ssDNA that is rapidly coated by RPA. Resected DSBs are substrates for MMEJ and HR, and the choice between these pathways is poorly understood. Polθ-helicase displaces RPA to promotes the synapsis of the opposing ends. If annealing occurs using internal microhomology, flaps are generated and are processed by FEN1. Polθ fills-in the gapped DNA and hands over the substrate to Lig3 to seal the end. When resected DNA is subject to long end-resection by EXO1/DNA2 and BLM, RPA1 is exchanged for Rad51 to promote strand invasion and copying from the sister chromatid.

Figure 2.. Overview of all reported Polθ activities.

Representation of the various activities that are carried out by Polθ. The major and well-established Polθ function is during DSB repair by MMEJ and has been studied using different substrates and in various model systems (green). Polθ has also been linked to the repair of breaks associated with replication forks (yellow). Lastly, Polθ employs its translesion synthesis activity to bypass UV-damaged bases and abasic sites (orange).

Figure 3.. Polθ is a unique multidomain enzyme.

Schematic representation of the different domains of human Polθ, depicting the structure and function of its helicase domain (pink), the unstructured central domain (grey) and the family-A polymerase domain (orange). The helicase domain drives MMEJ activity during chromosomal translocations, promotes Polθ dimerization, and suppresses HR and snap-back replication. The central domain contains three predicted RAD51 binding motifs that are not conserved in mouse. These motifs are implicated in suppression of HR through the inhibition of RAD51 nucleofilament formation. The primary and unequivocal function of the polymerase domain is to perform fill-in synthesis during MMEJ. The polymerase domain has also been reported to participate in tethering DNA ends. The polymerase domain comprised three insertion loops that are essential for Polθ to bypass bulky lesions and abasic sites, and were proposed to promote dimerization.