Human DNA polymerase θ grasps the primer terminus to mediate DNA repair

Abstract

DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Breast, lung and oral cancers over-express polymerase θ, and reduction of its activity in mammalian cells increases sensitivity to double-strand break inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ employs a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction to the 3’-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions, or extend poorly annealed DNA termini to mediate end-joining.

Essential Roles for Polymerase θ-Mediated End Joining in the Repair of Chromosome Breaks

“…Mre11 and XPF-ERCC1 are both attractive candidates for this nuclease activity, as they have appropriate substrate specificities and have already been implicated in Alt-NHEJ/MMEJ (Bennardo et al, 2008). The third step, synthesis from 3′ termini with 3- 6 bp annealed, is facilitated by the tight grasp Pol θ maintains on the primer termini (Zahn et al, 2015). Altogether, Pol θ is uniquely suited to direct this pathway.…”

Repair Pathway Choices and Consequences at the Double-Strand Break

“…This process allows pairing of microhomologies. Thereafter, Polυ can extend each strand from the base-paired region using the opposing overhang as a template (Figure 4) [83,84]. Interestingly, Polυ is upregulated in HR-deficient ovarian and breast cancers, suggesting that alt-EJ can serve as a backup pathway for the repair of DSBs when HR is defective.…”

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism

“…Structural analyses of Pol ν and Pol θ complexed with DNA substrate reveal potential mechanisms for low-fidelity TLS DNA synthesis and end-joining activities (23, 127). The overall structures of Pol ν and Pol θ ternary complexes are essentially identical to those of high-fidelity A-family members (as shown in Fig.…”

“…In solution Pol θ is more flexible and efficient in accommodating primer looping-out than Pol ν (23). Pol θ also has a series of positively charged residues in the thumb domain that strengthens its interactions with the primer strand (127). A combination of tight primer-strand binding and alternative template/primer alignment may underlie Pol θ-mediated end-joining.…”