Linking DNA polymerase theta structure and function in health and disease

Abstract

DNA polymerase theta (Pol θ) is an error-prone A-family polymerase that is highly conserved among multicellular eukaryotes and plays multiple roles in DNA repair and the regulation of genome integrity. Studies conducted in several model organisms have shown that Pol θ can be utilized during DNA interstrand crosslink repair and during alternative end-joining repair of double-strand breaks. Recent genetic and biochemical studies have begun to elucidate the unique structural features of Pol θ that promote alternative end-joining repair. Importantly, Pol θ-dependent end joining appears to be important for overall genome stability, as it affects chromosome translocation formation in murine and human cell lines. Pol θ has also been suggested to act as a modifier of replication timing in human cells, though the mechanism of action remains unknown. Pol θ is highly upregulated in a number of human cancer types, which could indicate that mutagenic Pol θ-dependent end joining is used during cancer cell proliferation. Here, we review the various roles of Pol θ across species and discuss how these roles may be relevant to cancer therapy.

Keywords: Carcinogenesis; Helicase; Homologous recombination; Indels; MMEJ; Translesion synthesis.

Fig. 1

Schematic of the domain structure of human Pol θ. a Domain structure of Pol θ. Domains include an N terminal helicase-like domain, a long unstructured central domain, and a C terminal polymerase domain. Within the polymerase domain is a non-functional exonuclease domain. b The polymerase domain of Pol θ contains finger, thumb, and palm subdomains. Insert 1 lies in the thumb domain while inserts 2 and 3 lie in the palm domain. The exonuclease subdomain contains 2 additional insertions, loop exo1 and loop exo2

Fig. 2

DNA double-strand break repair pathways. a Classical non-homologous end joining. A DNA break occurs during G1 phase. Ku70/Ku80 binds DNA ends and keeps them in close proximity (i). DNA-PKcs binds to Ku (ii) and recruits NHEJ core proteins including XRCC4 and DNA ligase 4. DNA ligase 4 ligates broken ends together (iii). b Homologous recombination. A DNA break occurs during S/G2. DNA is extensively resected in a 5′→3′ direction (i). The exposed 3′ single-stranded DNA is coated with RPA to stabilize it (ii). RPA is displaced by Rad51, with the help of Rad51 loading proteins (iii). Rad51 facilitates strand invasion and homology searching (iv). After DNA is copied from a homologous template, the D-loop is resolved (v). c Microhomology-mediated end joining. A DNA break occurs during S/G2. Limited resection occurs in a 5′→3′ direction (i). Microhomologies present at the DNA ends are aligned and stabilized by Pol θ, which then synthesizes DNA to fill in gaps (ii). DNA ligase 3/XRCC1 binds DNA to seal nicks (iii)

Fig. 3

Models of Pol θ-mediated MMEJ A double-strand break occurs (i) and DNA ends are resected (ii). Pol θ (green) aligns microhomologies (blue) located at the end of each ssDNA (iii). Pol θ synthesizes DNA to fill in the gap and strand displaces dsDNA, possibly aided by the helicase domain (purple) (iv). This repair process generates small deletions. Pol θ also aligns microhomologies that are located internally on ssDNA, leaving unpaired flaps (v). Flaps are cleaved by an endonuclease and Pol θ continues to synthesize DNA and displace dsDNA (vi). This process generates larger deletions. In the event that no microhomologies exist on ssDNA, Pol θ can utilize DNA overhangs as a template to generate microhomologies in “snap-back” synthesis, while displacing dsDNA (vii). Once microhomologies exist, they are aligned by Pol θ (viii) and Pol θ then fills in the gap (ix). This repair process generates templated insertions and deletions

Fig. 4

Mechanisms by which Pol θ might promote the initiation of MMEJ. a Once microhomologies are aligned, insert 2 may interact with the 5′ phosphate during DNA synthesis. This interaction could enhance binding ability of Pol θ and could also facilitate strand displacement during nascent DNA synthesis. b Insert 2 may also interact with the 3′ (n−1) phosphate on the nascent DNA strand. This interaction could facilitate nucleotide incorporation and stabilize mispaired nucleotides