A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair.

Patrick Herr,Bastiaan Evers,Daniel Ebner,Erik Sonnhammer,Guy Kingham,Cecilia Lundin,Oliver Frings,Oliver Mortusewicz,Timea Palmai-Pallag,Christina Bauerschmidt,Thomas Helleday

doi:10.1038/celldisc.2015.34

Abstract

To identify new regulators of homologous recombination repair, we carried out a genome-wide short-interfering RNA screen combined with ionizing irradiation using RAD51 foci formation as readout. All candidates were confirmed by independent short-interfering RNAs and validated in secondary assays like recombination repair activity and RPA foci formation. Network analysis of the top modifiers identified gene clusters involved in recombination repair as well as components of the ribosome, the proteasome and the spliceosome, which are known to be required for effective DNA repair. We identified and characterized the RNA polymerase II-associated protein CDC73/Parafibromin as a new player in recombination repair and show that it is critical for genomic stability. CDC73 interacts with components of the SCF/Cullin and INO80/NuA4 chromatin-remodeling complexes to promote Histone ubiquitination. Our findings indicate that CDC73 is involved in local chromatin decondensation at sites of DNA damage to promote DNA repair. This function of CDC73 is related to but independent of its role in transcriptional elongation.

Highlights

The DNA damage response is a safeguarding mechanism that ensures maintenance of the genomic integrity of cells
Genome-wide siRNA screen for regulators of HRR HRR is induced by two-ended double-strand breaks (DSBs) or by one-ended DSBs formed at collapsed replication forks, caused, for instance, by the topoisomerase I–inhibitor Camptothecin (CPT) [14], and different enzymes may be involved in the subsequent HRR
We found a high correlation between the genes involved in forming RAD51 foci following CPT and IR (Figure 1a), indicating either that the same proteins are involved in two-ended and one-ended HRR or alternatively that a similar substrate for HRR is formed after both IR- and CPT-induced lesions

Summary

Introduction

The DNA damage response is a safeguarding mechanism that ensures maintenance of the genomic integrity of cells. Aberrant DNA repair leads to genomic instability and cancer [1]. Two main pathways have been identified to repair DNA double-strand breaks (DSBs) in the cell. Non-homologous endjoining resolves DSBs by direct ligation of DNA ends. RNA-metabolizing enzymes have previously been identified to be involved in DNA damage response and HRR [3, 4]. Among many known HRR proteins, the top hit of our validation experiments was CDC73, which is encoded by the HRPT2 tumor suppressor gene. CDC73 was shown to be associated with the PAF1/RNA polymerase II transcriptional elongation complex [5]

Methods

Results

Conclusion