Essential role for polymerase specialization in cellular nonhomologous end joining

Abstract

Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) μ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol μ using the unpaired base adjacent to the downstream 5′ phosphate even when there are available template sites further upstream of this position; this makes Pol μ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol μ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.nonhomologous end joining | double-strand break repair | Pol X | polymerase mu | polymerase lambda

Creative template-dependent synthesis by human polymerase mu

“…To better understand the substrate preferences of Pol λ and Pol μ, here we present crystal structures of the human Pol μ (hPol μ) catalytic domain in complex with a 2-nt gapped DNA substrate, from DNA binding through nucleotide binding and incorporation. These structures are consistent with the in vivo NHEJ assays from Pryor et al (8) and support a "1-nt gap" spacing rule for Pol μ in which Pol μ prefers to engage all substrates in a similar fashion, using the 5ʹ unpaired template base in the gap as though it were a singlenucleotide (1-nt) gap. Additionally, this study provides insights into Pol μ substrate preference during in vitro polymerization reactions and how this enzyme might function during NHEJ in vivo.…”

“…In the context of DSB substrates lacking any sequence complementarity, namely substrates with an unpaired primer terminus, Pol μ uses 1-and 2-nt gaps equally well (figures 1B and 3A in ref. 8, the companion article). Interestingly, however, Pol μ appears to interact with short gaps of any length as though they were 1-nt gaps, preferentially filling the gap in a template-dependent manner, using the 5ʹ unpaired template base in the gap rather than the first available 3ʹ unpaired base proximal to the primer terminus, which results in deletion frameshifts.…”

“…As suggested by Pryor et al (figures 3A and 4B in ref. 8, the companion article), Pol μ addresses 2-nt gapped substrates by a single polymerization event, using the templating nucleotide at the 5ʹ end of the gap even when supplied with an excess of incoming nucleotide complementary to the 3ʹ templating nucleotide (up to 18-fold preference) (Fig. 1A).…”

“…Pol μ is the only known polymerase that can use noncomplementary substrates, such that in Pol μ knockout cells, these ends are largely unrepaired (figures 1B and 3A in ref. 8, the companion article). To better understand the substrate preferences of Pol λ and Pol μ, here we present crystal structures of the human Pol μ (hPol μ) catalytic domain in complex with a 2-nt gapped DNA substrate, from DNA binding through nucleotide binding and incorporation.…”

“…Recent work by Pryor et al (8) (companion article in this issue) has demonstrated a strong preference for Pol λ over Pol μ in repairing the majority of complementary DSBs. This is advantageous, because Pol λ displays higher fidelity of synthesis (9,10).…”