RNAi Screening Uncovers a Synthetic Sick Interaction between CtIP and the BARD1 Tumor Suppressor.

Hella A Bolck,Roger Meier,Simon F Nørrelykke,Antonio Porro,Alessandro A Sartori,Michael Stebler,Christina Zurfluh,Kay Hänggi,Christine Von Aesch,Sara Przetocka,Vincent Spegg,Matthias Altmeyer,Peter Horvath

doi:10.3390/cells11040643

Abstract

Human CtIP is best known for its role in DNA end resection to initiate DNA double-strand break repair by homologous recombination. Recently, CtIP has also been shown to protect reversed replication forks from nucleolytic degradation upon DNA replication stress. However, still little is known about the DNA damage response (DDR) networks that preserve genome integrity and sustain cell survival in the context of CtIP insufficiency. Here, to reveal such potential buffering relationships, we screened a DDR siRNA library in CtIP-deficient cells to identify candidate genes that induce synthetic sickness/lethality (SSL). Our analyses unveil a negative genetic interaction between CtIP and BARD1, the heterodimeric binding partner of BRCA1. We found that simultaneous disruption of CtIP and BARD1 triggers enhanced apoptosis due to persistent replication stress-induced DNA lesions giving rise to chromosomal abnormalities. Moreover, we observed that the genetic interaction between CtIP and BARD1 occurs independently of the BRCA1-BARD1 complex formation and might be, therefore, therapeutical relevant for the treatment of BRCA-defective tumors.

Highlights

Faithful transmission of genetic information to daughter cells is a central process for the maintenance of genome stability and the suppression of cancer and relies on accurate and complete DNA replication during S-phase
We have recently found that CtIP and BRCA1, two Homologous recombination (HR) factors, synergize to preserve genome stability upon replication stress [9]
BRCA1-associated RING domain protein 1 (BARD1) was among the strongest hits (RSA p-value 0.0175), with all three independent siRNAs used in the screen reproducibly reducing cell growth in a CtIP-deficient background, as denoted by negative epsilon scores (Figure 1C and Figure S1C)

Summary

Introduction

Faithful transmission of genetic information to daughter cells is a central process for the maintenance of genome stability and the suppression of cancer and relies on accurate and complete DNA replication during S-phase. Proteins commonly involved in homology-directed repair (HDR) of DNA double-strand breaks (DSBs) protect stalled replication forks [3]. There is ample evidence that loss of BRCA1 and BRCA2 results in nucleolytic degradation of nascent DNA at stalled forks, giving rise to chromosomal instability [4]. More recent findings described additional functions of CtIP in response to replication stress, including efficient replication fork restart, suppression of new origin firing and promoting common fragile site stability [6,7]. We have recently proposed that CtIP limits excessive fork degradation in a BRCA1-independent manner [9]. This was rather unexpected as a direct interaction between the tandem BRCT repeats of BRCA1 and phosphorylated CtIP was reported to promote efficient DSB resection and subsequent HDR [10–13]. Several studies have challenged this notion suggesting that complex formation with BRCA1 is largely dispensable for CtIP-mediated resection and ensuing HDR [14–16]

Methods

Results

Discussion

Conclusion