Genome Protection by DNA Polymerase θ

Abstract

DNA polymerase θ (Pol θ) is a DNA repair enzyme widely conserved in animals and plants. Pol θ uses short DNA sequence homologies to initiate repair of double-strand breaks by theta-mediated end joining. The DNA polymerase domain of Pol θ is at the C terminus and is connected to an N-terminal DNA helicase-like domain by a central linker. Pol θ is crucial for maintenance of damaged genomes during development, protects DNA against extensive deletions, and limits loss of heterozygosity. The cost of using Pol θ for genome protection is that a few nucleotides are usually deleted or added at the repair site. Inactivation of Pol θ often enhances the sensitivity of cells to DNA strand-breaking chemicals and radiation. Since some homologous recombination-defective cancers depend on Pol θ for growth, inhibitors of Pol θ may be useful in treating such tumors.

Keywords: DNA double-strand breaks; DNA helicase; DNA polymerases; DNA repair; mutations; translesion DNA synthesis.

Figure 1

DNA polymerase θ three-domain architecture and depiction of the human protein, encoding a 2,590–amino acid polypeptide. The helicase-like domain includes five main structural elements (77): two RecA-like subdomains (RecA1 and RecA2), a winged helix subdomain (WH), a helical bundle, and a helix-hairpin-helix subdomain (HhH). The central region is predicted to be mostly disordered (12, 95) and has a variable length between organisms. The polymerase domain consists of an A-family DNA polymerase with a nonfunctional editing exonuclease domain (Exo) and the fingers (F), palm (P), and thumb (T) regions characteristic of DNA polymerases (124).

Figure 2

Three major strategies for repair of a DNA double-strand break. In nonhomologous end joining (NHEJ, left), the ends of the break are bound and protected by Ku protein homologs, and then reactions proceed to join the ends, often involving limited nuclease trimming and DNA synthesis to repair broken DNA before ligation. Alternatively, 5′ ends at the break may be processed by nuclease-mediated resection. Resection generates 3′ single-stranded DNA tails. In the process of homologous recombination repair (HR, middle), a 3′ tail pairs with an intact sister strand in a recombination process dependent on Rad51. The primary HR repair pathway is synthesis-dependent strand annealing in which one strand is extended for some distance by DNA synthesis so that it can be paired with its original partner and repair completed. In some cases, 3′ ends are joined by theta-mediated end joining (TMEJ, right). Pol θ locates a microhomology, typically of 2–6 base pairs in the 3′ single strands. Processing of the 3′ ends of the DNA will usually have to occur to remove unpaired regions and allow completion of synthesis (see Figure 4). In general, TMEJ creates short deletions or templated insertions in the DNA (red box).

Figure 3

Pol θ makes unique contacts that grasp the primer strand and the incoming nucleotide. A view of the ternary complex of the polymerase domain bound to furan-containing DNA and ddATP (PDB ID 4X0P) (126). The finger, palm, thumb, and insert 2 regions are depicted in blue, red, green, and yellow, respectively. The DNA backbones of the template and primer strands are shown in pale yellow and orange, respectively, with the 3′ terminus becoming red. The divalent cation (metal A) coordinated in the polymerase active site is depicted as a magenta sphere. Of the five positively charged residues contacting the primer backbone, two contacts are conserved in all A-family polymerases (shown with white carbons), and three of them are unique residues in Pol θ (cyan carbons), with Arg2254 emerging from the distinctive insert 2 (yellow). Abbreviations: ddATP, 2′,3′-dideoxyadenosine-5′-triphosphate; PDB ID, Protein Data Bank identification; Pol θ, DNA polymerase θ.

Figure 4

The task of TMEJ is to join a break by making use of microhomologies within single-stranded, 3′-ended DNA tails. In the general case, preexisting microhomologies are internal. If one or both tails can be removed by a nuclease so that Pol θ can extend from a microhomology, as shown in the box at the right, then repair could be completed. Another route is shown in the box at the left. Pol θ can manipulate 3′ single-stranded DNA by engaging in one or more short tracts of inter- or intramolecular synthesis, usually using nearby DNA as a template. This produces the templated insertions that are characteristic of TMEJ. A terminus is generated with a sequence different from the original, allowing iterative microhomology searches. In principle, the immediate extension of only one 3′ end is sufficient to generate stable joining of the break. A single remaining 3′ tail could then be removed by a nuclease after the DNA polymerase has dissociated, at which point further extension and sealing of the break by a DNA ligase can occur. Abbreviation: TMEJ, theta-mediated end joining.