The role of the CNOT complex in the mammalian DNA damage response

The cellular genome is subject to continuous attack, resulting in the occurrence of tens of thousands of lesions in the DNA per cell per day. To counteract this, a complex series of pathways, collectively known as the DNA damage response (DDR), has evolved and this is able to detect damage and, if possible, repair the DNA. The inability to correct this damage is a major underlying cause of cancers and neurodegeneration as well as other diseases. DNA damage can take a number of forms: single and double strand breaks, the formation of bulky adducts and alkylated bases as well as mismatched bases. These can result from different damaging agents and DNA replication stress. Specific pathways have evolved to deal with each of these types of lesion. Double strand breaks (DSBs) in the cellular DNA are a relatively rare event, compared to other forms of damage, but tend to be toxic to the cell if left unrepaired; in addition they can give rise to chromosomal rearrangements and genomic instability. DSB repair pathways are based on the activities of three related kinases-Ataxia Telangiectasia mutated (ATM), ATM and Rad3-related (ATR) and DNA dependent protein kinase (DNA-PK). Following detection of the DNA damage, one or more of the kinases are activated leading to phosphorylation of multiple downstream targets and repair of the lesion if the damage is not too great. The DDR pathways comprise a large number of components, with, simplistically, specific proteins involved in homologous recombination, non-homologous end joining and single strand break repair amongst others. We have recently shown that certain components of the CNOT complex (a large multi-functional complex well-characterized in yeast but much less so in mammalian cells) play a role in the response to DNA damage. Activities associated with proteins in the complex include deadenylase, ubiquitin E3 ligase, transcriptional regulation and RNA metabolism functions. In yeast loss of various CCR4NOT (the nomenclature for the yeast complex) proteins sensitises cells to different forms of DNA damage although details of the pathways involved remain unclear; even less information is available on a possible DDR role in mammalian cells. To date our main focus has been on a little-characterised CNOT component which doesn’t have a yeast equivalent-tankyrase1-binding protein 1 (TNKS1BP1, also known as Tab182). We have shown that loss of Tab182 sensitises cells to ionising and UV radiation as well as nucleotide depletion with hydroxyurea (HU). Significantly, it has also been shown that cells deficient in Tab182 and other CNOT proteins are hypersensitive to agents which induce DNA replication stress and exhibit abnormal replication dynamics.

The aims of this project are to use siRNAs to deplete different components of the complex (of which there are about a dozen) and determine which ones affect the cellular responses to different forms of DNA damage and DNA replication stress. In a more detailed study CRISPR/cas9 gene editing will be used to engineer one or more cell lines in which the most significant (from a DDR point of view) CNOT genes are knocked-out. The effects of this on the different DNA repair pathways in these lines will analysed in detail. We will pay particular attention to DNA replication stress where we have already shown that Tab182 depletion causes marked abnormalities. In a second element of the project the effects of different DNA damaging agents on CNOT enzymatic activities (for example, deadenylation of RNA and ubiquitin E3 ligase activities) will be determined.