Artemis is required to improve the accuracy of repair of double-strand breaks with 5′-blocked termini generated from non-DSB-clustered lesions

Clustered DNA lesions are defined as ≥2 damage events within 20bp. Oxidised bases, abasic (AP) sites, single-strand breaks and double-strand breaks (DSBs) exist in radiation-induced clusters, and these lesions are more difficult to repair and can be more mutagenic than single lesions. Understanding clustered lesion repair is therefore important for the design of complementary treatments to enhance radiotherapy. Non-DSB-clustered lesions consisting of opposing AP sites can be converted to DSBs by base excision repair, and non-homologous end-joining (NHEJ) plays a role in repairing these DSBs. Artemis is an endonuclease that removes blocking groups from DSB termini during NHEJ. Hence, we hypothesised that Artemis plays a role in the processing of DSBs or complex DSBs generated from non-DSB-clustered lesions. We examined the repair of clusters containing two or three lesions in wild-type (WT) or Artemis-deficient (ART−/−) mouse fibroblasts using a reporter plasmid. Each cluster contained two opposing tetrahydrofurans (an AP site analogue), which AP endonuclease can convert to a DSB with blocked 5′ termini. Loss of Artemis did not decrease plasmid survival, but did result in more mutagenic repair with plasmids containing larger deletions. This increase in deletions did not occur with ClaI-linearised plasmid. Since Mre11 has been implicated in deletional NHEJ, we used small interfering RNA to reduce Mre11 in WT and ART−/− cells, but decreasing Mre11 did not change the size of deletions in the repair products. This work implicates Artemis in limiting the deletions introduced during repair of 5′-blocked termini DSBs generated from non-DSB-clustered lesions. Decreasing repair accuracy without decreasing repair capacity could result in mutated cells surviving irradiation. Inhibiting Artemis in normal cells could promote carcinogenesis, while in tumour cells enhanced mutagenic repair following irradiation could promote tumour recurrence.