Integrator complex subunit 6 (INTS-6) mediates DNA damage response in Caenorhabditis elegans

Abstract

The Caenorhabditis elegans Integrator complex is a set of at least 13 evolutionarily conserved proteins that binds the C-terminal domain of RNA polymerase II to regulate snRNA 3'-end processing and gene expression. Here we show that the Integrator subunit 6 intervenes in the DNA damage response in C. elegans . We find that upon X-ray radiation, INTS-6 is necessary for RAD-51 foci formation. In addition, CDK-1 Tyr-15 phosphorylation depends on the presence of INTS-6 . This work adds a new piece to elucidate the Integrator complex mechanism of action in DNA repair.

Figure 1. Integrator complex subunit 6 (INTS-6) mediates DNA damage response in Caenorhabditis elegans

(A) INTS-6-associated proteins identified by anti-FLAG affinity purification. In addition to members of the Integrator complex, other proteins were immunoprecipitated along with INTS-6::3xFLAG::GFP, including some related to DNA damage response, such as LAF-1 and NABP-1 (adapted from Gomez-Orte et al., 2019). (B) Immunofluorescence images of RAD-51 foci formed after ionizing radiation. ints-6 knockdown impairs RAD-51 recruitment to DSBs following IR. As shown in the pictures, there are no RAD-51 foci before IR, either in worms fed the L4440 bacterial RNAi clone (panel 1, 5, 9) or fed the ints-6 bacterial RNAi clone (panel 2, 6, 10). Following irradiation, RAD-51 foci can be observed in the gonads of worms treated with the L4440 bacterial RNAi clone (panel 3, 7, 11) but no RAD-51 foci are observed in the mitotic region of gonads of worms treated with the bacterial RNAi clone of ints-6 (panel 4, 8, 12). Scale bar: 10 μm. Immunofluorescence images of phosphorylated CDK-1. ints-6 knockdown abrogates Tyr15 CDK-1 phosphorylation in response to DNA damage. Tyr15 CDK-1 phosphorylation was detected in the nuclei of control gonads after irradiation (panel 15,19,23). However, in the gonads of worms knocked down for ints-6 , phosphorylation of Tyr15 CDK-1 following IR was not detected (panel 16, 20, 24). Scale bar: 10 μm. (C) Proposed model. Upon the occurrence of a double strand break in DNA, the MRN complex together with ATM senses it. MRN then activates ATM triggering the DNA damage response pathway. One of the targets of ATM is hSSB1, which is phosphorylated and mobilized to the site of damage. It interacts directly with MRN complex stimulating its recruitment to the break site. The DNA end resection is then initiated, creating single stranded DNA overhangs. Based on our results and others previously published, we believe that at this point INTS-6 acts. These ssDNA ends are then coated with the RPA protein. Finally, RPA is replaced by RAD-51 and the resulting RAD-51 coated filament performs homology search and strand invasion, allowing DNA synthesis at the resected strand and subsequent repair.