Studying the causes and consequences of DNA damage on the molecular and cellular level is a major topic of research within cancer biology. DNA damage not only causes cancer, but is also used as a means to cure cancer through radio- or chemotherapy. It is also responsible for the side effects of these treatments. DNA double-strand breaks (DSBs) are the most cytotoxic lesions induced by ionizing radiation and certain anti-cancer drugs, and appropriate signaling and repair of DSBs are therefore of prime importance.

The main focus of research in our laboratory is to better understand how human cells respond to DNA damage and maintain genomic integrity - an important factor in the etiology of cancer. We are particularly interested in the repair of DSBs. Because DSBs are the most dangerous lesions a cell can encounter, detailed knowledge of the factors participating in their repair and in the regulation of this process is crucial if we are to improve current cancer therapy and suggest novel strategies to fight this disease.

We are deploying several approaches to achieve our aim. Besides the exciting prospect of identifying novel factors involved in DSB repair by high-throughput proteomic screens, we use a combination of biochemistry, cell biology and genetics to gain novel insights into the molecular mechanisms of this very complex repair network and its regulation by post-translational modifications such as phosphorylation and ubiquitylation. DSBs are repaired by two evolutionarily conserved mechanisms: homologous recombination (HR) and non-homologous end-joining (NHEJ). However, the criteria that decide which pathway repairs which DSB remain largely unknown and elucidating how the choice is regulated is another major topic in our laboratory.

To preserve genomic integrity and aid survival, DSBs alarm the cellular DNA damage response machinery, a multifaceted process orchestrated in mammalian cells by the ATM and ATR kinase signaling pathways. In response to genotoxic insults, ATM and ATR phosphorylate key substrates involved in DNA repair and cell cycle control. In S and G2 phases of the cell cycle, DSBs are resected to produce single-stranded DNA that contribute to cell cycle checkpoint activation and trigger repair by HR. DNA end resection, a process that is still not understood in great detail, has been shown to be dependent on both ATM and CDK activities, but the targets of these kinases that are involved in DNA resection have not yet been identified.

The identification of novel factors implicated in the DNA damage response by high-throughput screens

Human CtIP (RBBP8) was originally discovered as a cofactor of the transcriptional co-repressor CtBP. In addition to transcriptional regulation, CtIP plays a crucial role in the repair of DNA double-strand breaks (DSBs) by initiating homologous recombination. Furthermore, CtIP has been shown to interact with two tumor suppressor proteins: retinoblastoma (pRB) and BRCA1. Recent evidence suggests, that CtIP is able to counteract pRB-mediated G1 arrest while the CtIP-BRCA1 complex is important to facilitate DSB resection and subsequent repair during S and G2 phase. Genetic studies in mice revealed that CtIP is an essential gene in mammalian cells. Homozygous CtIP-/- mice were inviable, while haploid insufficiency predisposed mice to multiple types of tumors, indicating that CtIP might itself be a tumor suppressor. Thus, CtIP emerges as a multivalent adaptor connecting cellular path­ways such as cell cycle checkpoint control, transcriptional regulation and tumor suppression, key events known to be strongly implicated in tumorigenesis and tumor progression. In order to further expand our understanding of the function(s) of CtIP in various biological pathways and to uncover new therapeutical approaches to treat cancer, we plan to conduct multiple systematic RNAi screens. Specifically, we aim to interrogate a large number of human genes for synthetic genetic interactions (synthetic lethality or rescue) with CtIP.

Figure 1. CtIP is required for DNA end resection.

Sensing of DSBs by MRN leads to the activation of ATM and subsequent phosphorylation of several downstream targets involved in DNA damage response (DDR) such as H2AX and CHK2. Initial DNA end resection is realized through MRN and CtIP, followed by extensive resection carried out by EXO1. The resulting 3’ssDNA overhangs are immediately coated by RPA. As a consequence, ATR is recruited and hyperphosphorylates more DDR target proteins including RPA2 and CHK1 required for G2/M checkpoint activation. Finally, RPA is exchanged for RAD51 and the RAD51-ssDNA nucleoprotein filaments initiates strand invasion and HR repair with the help of additional factors.

  The regulation of DSB repair by post-translational modifications

Human CtIP is involved in the DNA damage response by promoting DNA end resection which is required for the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). Several recent studies have indicated that CtIP is under tight regulation by a number of post-translational modifications, including phosphorylation and ubiquitylation. However, while it was established that phosphorylation by CDK is required for DSB resection, it is still largely unknown how ubiquitylation and deubiquitylation controls CtIP function. For instance, it has been reported that CtIP polyubiquitylation by the BRCA1/BARD1 E3 ubiquitin ligase does not target CtIP for degradation but, instead, triggers CtIP association with chromatin following DNA damage. Besides BRCA1, a yeast-two hybrid revealed interaction of CtIP with SIAH-1, another E3 ubiquitin ligase but this study did not address whether SIAH-1 triggers CtIP ubiquitylation and subsequent degradation. It has been shown that CtIP protein levels peak in S/G2 phase while its transcript levels remain constant throughout the cell cycle, suggesting that CtIP is regulated by the ubiquitin-proteasome pathway. In agreement with this hypothesis, we did observe a significant increase of CtIP protein levels upon treatment of cells with MG-132. Furthermore, using mass spectrometry-based proteomic screens, we have identified HECT- and RING-domain-containing E3 ubiquitin ligases as potentially novel CtIP interacting partners. In addition, we have also identified a ubiquitin hydrolase, indicative of a dynamic balance between CtIP ubiquitylation and deubiquitylation. We are now in the process of verifying these hits. Ultimately, our goal is to identify the cellular pathways promoting CtIP ubiquitylation and to understand its physiological relevance.

Figure 2. Hypothetical model: how PIN1-mediated CtIP isomerization controls DNA end resection. During S/G2, CtIP together with other nucleases promotes the resection of DSBs. Following resection initiation, proline-directed kinases including CDK2 phosphorylate CtIP on T315 and S276, resulting in the binding of PIN1 to CtIP. PIN1-mediated isomerization of CtIP leads to CtIP ubiquitylation through an as yet unknown E3 ubiquitin ligase and subsequent CtIP degradation by the proteasome. This mechanism ensures an appropriate usage of DSB-end resection. Consequently, cells with abrogated PIN1 function or inherently low PIN1 levels display reduced NHEJ and aberrant (error-prone) forms of homology-directed repair due to enhanced CtIP resection activity (Hyperresection). In contrast, cells overexpressing PIN1 display reduced HR and increased NHEJ due to decreased CtIP resection activity (Hyporesection). Therefore, we propose that PIN1 plays an important role in the regulation of DSB repair particularly in late S and G2 phases of the cell cycle.

Discovering novel connections between CtIP and genome other genome surveillance pathways

DNA double-strand breaks (DSBs) are one of the most critical lesions with respect to survival and preservation of genomic integrity. A key role in recognizing, signaling and repair of DSBs in mammalian cells is ascribed to the MRE11-RAD50-NBS1 (MRN) complex. Our study is aimed to gain more mechanistic insights on the role of RAD50 in the DNA damage response, which so far has been mostly attributed to serve as a scaffolding component of the MRN complex. To gain more detailed mechanistic insights into how RAD50 contributes to the maintenance of genome integrity, we are analyzing biochemical and cellular properties of human RAD50S (‘Separation-of-function’) mutations. More than 20 years ago, several rad50S alleles were isolated and characterized in S. cerevisiae. These alleles conferred no overt MMS sensitivity to the yeast cells but still blocked viable spore formation, indicative for a strong defect in meiosis. To reveal the underlying mechanism resulting in increased hypersensitivity of RAD50S to CPT, we are currently addressing potential repair and/or cell cycle checkpoint defects in established RAD50S-expressing human cell lines. In summary, our detailed characterization of RAD50S phenotypes using isogenic human cell lines should eventually lead to a better understanding of the function of the MRN complex in the maintenance of genome stability. Fanconi anemia (FA) is a rare hereditary disorder characterized by bone marrow failure, multiple congenital abnormalities and increased susceptibility to cancer. Cells isolated from FA patients display chromosomal instability and hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents such as mitomycin C (MMC) and cisplatin. ICLs represent highly toxic DNA lesions that prevent transcription and replication by inhibiting DNA strand separation. Recent studies indicate that FA pathway orchestrates ICL repair mediated by nucleotide excision repair (NER), translesion synthesis (TLS) and, in a final step, homologous recombination (HR). CtIP is required for normal embryonic development and promotes the resection of DSBs during HR. Thus, it is generally believed that the function of CtIP in ICL repair is through DNA end resection, downstream of the initial ICL processing step and genetically distinct from the FA pathway. Our current work is focused on the potential interplay between CtIP and the FA pathway in the repair of ICLs.

The role of CtIP in tumorigenesis and cancer

Since its discovery more than 10 years ago as an interacting protein of CtBP, RB, and BRCA1, human CtIP has emerged as a polyvalent adaptor protein involved in the regulation of transcription and cell cycle checkpoints. Based on the partnership with these known tumorsuppressors, CtIP has been postulated to be a candidate tumor susceptibility gene itself. Support for this hypothesis came with the observation that Ctip+/- heterozygous mice develop multiple types of tumors, predominantly large B-cell lymphomas, while homozygous deletion of Ctip results in early embryonic lethality. Moreover, CtIP cooperates with MRN in the initial processing of DSBs, called DNA end resection, which is required for homologous recombination. We have provided evidence that CtIP-dependent DNA end resection may actively suppresses non-homologous end-joining (NHEJ), the second major DSB repair pathway in human cells, which simply rejoins DSB ends. Faithful repair of DSBs is crucial for the maintenance of genomic stability, as improper repair can lead to chromosomal rearrangements such as translocations. Reciprocal chromosomal translocations are implicated in the etiology of many hematologic tumors, particularly in B-cell lymphomas. The result is either the deregulation of a proto-oncogene, or the expression of a novel fusion protein with oncogenic potential. However, which DSB repair pathway gives rise to translocations and under which conditions is still an area of intensive research. In this project we would like to investigate a potential role of CtIP in the events leading to translocations and, in the first phase, to concentrate our analysis on the function of CtIP in DSB repair in Burkitt’s lymphoma. Based on our findings, we hope to be able to improve predictions of the clinical outcome of current chemotherapeutic regimens in lymphomas. Moreover, data from our ongoing biochemical characterization of CtIP might reveal new avenues leading towards the development of novel therapeutic strategies in the treatment of some specific forms of lymphoma.

Our projects are funded by the Vontobel Foundation, the Swiss National Science Foundation, the Promedica Foundation, the Zurich Cancer League, the Swiss Cancer League, the Olga Mayenfisch Foundation, Sophien Foundation and the ‘Forschungskredit’ of the University of Zurich.