DNA Polymerase θ: A Unique Multifunctional End-Joining Machine

Abstract

The gene encoding DNA polymerase θ (Polθ) was discovered over ten years ago as having a role in suppressing genome instability in mammalian cells. Studies have now clearly documented an essential function for this unique A-family polymerase in the double-strand break (DSB) repair pathway alternative end-joining (alt-EJ), also known as microhomology-mediated end-joining (MMEJ), in metazoans. Biochemical and cellular studies show that Polθ exhibits a unique ability to perform alt-EJ and during this process the polymerase generates insertion mutations due to its robust terminal transferase activity which involves template-dependent and independent modes of DNA synthesis. Intriguingly, the POLQ gene also encodes for a conserved superfamily 2 Hel308-type ATP-dependent helicase domain which likely assists in alt-EJ and was reported to suppress homologous recombination (HR) via its anti-recombinase activity. Here, we review our current knowledge of Polθ-mediated end-joining, the specific activities of the polymerase and helicase domains, and put into perspective how this multifunctional enzyme promotes alt-EJ repair of DSBs formed during S and G2 cell cycle phases.

Keywords: DNA polymerase; DNA repair; alternative end-joining; cancer; genome instability; microhomology-mediated end-joining; replication repair.

Figure 1

Schematic representation of Polθ and homologous proteins. The helicase, central and polymerase domains of POLQ are depicted at the top. The superfamily 2 (SF2) helicase domain contains a conserved nucleotide binding site (NT, dark grey), a conserved DEAH box motif (DEAH, dark grey), and a conserved helicase C-terminal domain (Helicase-C, dark grey). RAD51 binding domains (black) are found in the helicase and central domain. The polymerase domain contains a conserved A-family polymerase subdomain (blue), an inactive 3’–5’ exonuclease-like subdomain (red), and three unique insertion loops (green). Select POLQ polymerase and helicase orthologs are illustrated with their respective sequence similarities to POLQ. Relative homolog positions are based on sequence alignments. Characteristic regions found in helicase homologs are a RNA polymerase II subunit A binding subdomain (POLR2A, purple), a scaffolding protein involved in DNA repair (SPIDR, orange), and a helicase RNaseD C-terminal domain (HRDC, brown). Percent sequence similarities between homologous proteins and either Polθ-polymerase or Polθ-helicase domains are indicated. Sequence alignments and similarities were determined using Clustal Omega.

Figure 2

Models of Polθ-mediated end-joining. Polθ-mediated end-joining requires 5’–3’ DNA resection performed by the Mre11-Rad50-Nbs1 (MRN) nuclease complex along with CtIP which results in 3’ ssDNA overhangs. Poly (ADP ribose) polymerase I (PARP1) promotes the recruitment of Polθ to double-strand breaks (DSBs) by an undefined mechanism. Polθ-mediated end-joining may be distinguished by two different mechanisms: microhomology-mediated (right), and microhomology-independent (left). The presence of substantial microhomology (≥3 bp; red highlighted sequence) is likely to increase the half-life of the DNA synapse and therefore promote the Polθ-mediated end-joining reaction (right). The absence of substantial microhomology (≤2 bp) is likely to significantly reduce the efficiency of the end-joining reaction due to a shorter half-life of the DNA synapse (left). Following DNA synapse formation, Polθ (orange) utilizes the opposing ssDNA overhang as a template in trans to extend the DNA, which stabilizes the end-joining intermediate. Polθ may then extend the other overhang in the opposite direction, which would further stabilize the end-joining intermediate. Nucleases (blue) are likely to be required for further processing of the DNA by trimming unannealed ssDNA ends. Last, Lig1 or Lig3 is required to seal the processed DNA end-joining intermediate.

Figure 3

Structure of the Polθ polymerase domain. (A) Crystal structure of the Polθ polymerase domain in complex with a primer-template (PDB code 4X0P). Palm (purple), fingers (cyan) and thumb (green) domains are indicated. The location and reported functional roles of insertion loops, which are unstructured in the crystal, are indicated. (B) Close up structure highlighting the primer (grey), loop 2 (green), and conserved positively charged residues (blue: R2202, R2254) that bind the 3’ terminus of the primer (PDB code 4X0P).

Figure 4

Model of Polθ-polymerase terminal transferase activity during end-joining. Schematic representation of Polθ-polymerase switching between three different terminal transferase activities during end-joining. Polθ-polymerase performs non-templated ssDNA extension, templated extension in cis, and templated extension in trans, and spontaneously switches between these activities during end-joining which results in a combination of templated and non-templated nucleotide insertions at repair junctions.

Figure 5

Structure of the Polθ helicase domain. (A) Crystal structure of Polθ-helicase (PDB code 5AGA). The location of helicase domains D1–D5 and AMP-PNP are indicated. (B) Close up structure of the conserved nucleotide binding domain in complex with AMP-PNP (PDB code 5AGA). (C) Structure of the tetrameric form of Polθ-helicase. Individual Polθ-helicase monomers are represented in blue, green, red and cyan (PDB code 5A9J). Symmetry axis is indicated as blue lines. (D) Close up structure of the RAD51 binding site (residues 861–868) (PDB code 5A9J).

Figure 6

Models of Polθ-helicase activity during end-joining. (A) Model of end-joining involving Polθ-helicase ssDNA annealing activity. Polθ-helicase may interact with RPA and promote ssDNA annealing at microhomologous regions which is likely to facilitate end-joining. After annealing and DNA synapse formation, the polymerase domain performs ssDNA extension by utilizing the opposing overhang as a template in trans. (B) Model of end-joining involving Polθ-helicase anti-recombinase activity. Alt-EJ and HR share the same resection mechanism and therefore compete during S and G2 cell-cycle phases. Polθ-helicase may dissociate RAD51 filaments, then perform annealing which would enable Polθ-polymerase extension and subsequent steps of end-joining.

Figure 7

Model of Polθ-mediated end-joining of arrested replication forks. Model of Polθ-mediated end-joining of replication forks arrested at a G-quadruplex (G4). Following the arrest of divergent replication forks at G4, nucleases may cleave the lagging strands which would generate DSBs with 3’ ssDNA overhangs. Polθ may then promote DSB repair via alt-EJ (left). Gap filling on the leading strands may leave the G4 intact (right).