The DNA within our cells is under constant attack by exogenously- and endogenously-arising DNA-damaging agents. The DNA-damage response (DDR) has evolved to optimise cell survival following DNA damage; it involves the recruitment of DNA repair proteins to sites of damage and the “checkpoint” events that slow down or arrest cell-cycle progression. Importantly, DDR proteins play key roles in preventing cancer, and their activities, in part, determine the outcome of cancer radiotherapy and chemotherapy.
Our particular focus is the detection and repair of DNA double strand breaks (DSBs). These are among the most dangerous lesions that can occur in the genome of eukaryotic cells. Proper repair of chromosomal DSBs is critical for maintaining cellular viability and genomic integrity and, in multicellular organisms, for suppression of mutagenesis.
Our work aims to decipher how – at the molecular level – cells detect DNA-damage then trigger the myriad events of the DDR. We also study how DDR proteins control other processes such as telomeric integrity. Towards this end, we are using a broad range of techniques and approaches, in both mammalian and yeast cells.
DNA double-strand breaks are repaired by two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR), Critchlow and Jackson, 1998. NHEJ is the major DNA repair mechanism in mammalian cells whereas HR is the predominant repair mechanism in budding yeast. Research in this lab has focussed on characterising a number of the key proteins involved in repair of DSBs via NHEJ and, in fact, it was Steve’s identification of DNA-dependent protein kinase (DNA-PK) whilst a postdoc in the US that first led to his interest in DNA repair. The NHEJ pathway in mammals requires DNA-PK, a multi-subunit protein comprising three components: Ku70 and Ku80 which together form a DNA end-binding complex (Ku), and the DNA-PKcs catalytic subunit. The Ku proteins bind DNA ends of DSBs and recruit other NHEJ factors such as DNA-PKcs to these ends, leading to activation of DNA-PKcs. Other proteins such as the Mre11-Rad50-Nbs1 (MRN) complex are required to process broken ends making them available for ligation by DNA ligase IV and XRCC4.
Following on from the identification of several of the key proteins involved in NHEJ both in mammalian cells and in yeast we have gone on to analyze the structural basis of their interactions and their functional consequences for DNA repair (reviewed in Doherty and Jackson, 2001).
Current work includes continuing to analyze the structure and function of Ku, elucidating the proteins involved in NHEJ in yeast and looking for other novel NHEJ factors both in mammalian cells and yeast.
One key component of both NHEJ and HR pathways is the Mre11-Rad50-Nbs1 (MRN) protein complex (Mre11-Rad50-Xrs2 in yeast). This complex has both exonuclease and endonuclease activities and is involved in processing the DNA ends at DSBs prior to religation in NHEJ and in DNA end-resection to create single strand DNA ends prior to homologous pairing and strand invasion in HR. The MRN complex is also involved in sensing DNA DSBs, described below.
Sensing and responding to DSBs
The first step in the cellular response to damage is sensing the lesions. In mammalian cells DSBs are detected by several factors including the MRN complex, Ku and three related proteins: DNA-PKcs, ATM and ATR. These latter three proteins are all members of the PIKK family of protein kinases and are able to signal the presence of DNA damage to the cell via phosphorylation of downstream target proteins. Mammalian ATM is involved primarily in sensing and responding to DSBs generated by agents such as IR. By contrast, ATR responds to a wider range of lesions. Once activated, the checkpoint PIKKs phosphorylate a range of factors including the checkpoint kinases CHK1 and CHK2 that then target effector proteins involved in modulating DNA repair, transcription and cell-cycle control.
Detection and signalling of DNA DSBs (image taken from Jackson and Bartek, 2009).
In addition to studying proteins that are able to sense DNA damage, our lab, and others, have identified novel proteins that act as adaptors or facilitators of the DNA damage response and are required for efficient activation and signalling of the DNA damage. For example, we identified the yeast protein Lcd1p that recruits Mec1p (the yeast homologue of ATR) to sites of DNA damage (Rouse and Jackson, 2002). More recently, we identified a novel human protein, MDC1, that is a binding partner of the MRN complex, recruiting the MRN complex to sites of DNA damage and acting as a mediator in the initial detection and processing of DNA damage (Goldberg et al., 2003).
More recently, we have identified a common evolutionarily conserved and functionally related motif in the C terminus of the PIKK partner proteins Nbs1, ATRIP and Ku80 that is required for their interaction with their respective PIKK, ATM, ATR or DNA-PKcs. These motifs are essential for the recruitment of these PIKKs to sites of DNA damage and for downstream PIKK-dependent signalling events that lead to cell cycle checkpoint arrest and the efficient repair of damaged DNA (Falck et al., 2005).
We are currently investigating how these different proteins interact, function and are regulated in order to modulate the DNA damage response.
Chromatin and the DNA damage response
In eukaryotic cells, DNA is densely packaged into chromatin. In order, therefore, for DSBs to be repaired efficiently there must be restructuring of the DNA to allow repair proteins access to the sites of DNA damage. For example we have shown that phosphorylation of the core histone H2A is required for efficient DSB repair (Downs et al., 2000). Furthermore, we have demonstrated that this phosphorylation event creates a ‘mark’ on the chromatin around the site of DNA damage that results in the binding of chromatin remodelling factors (Downs et al., 2004, Jazayeri et al., 2004).
We are continuing to investigate how DNA damage responses are influenced by chromatin structure in both mammalian cells and in yeast.
Telomeres, the structures at the ends of chromosomes, are not normally recognized by the DNA repair machinery. It is now known that a number of proteins involved in the DNA damage response including Ku physically associate with telomeres and actually play important roles in regulating normal telomeric functions (reviewed in d’Adda di Fagagna et al., 2004). We have established that DNA damage responses are triggered by short telomeres in human cells undergoing replicative senescence, and that the senescent state requires the continued actions of DNA damage checkpoint kinases (d’Adda di Fagagna et al., 2003). These findings have important implications for cancer – where cells generally escape senescence – and other age-related diseases.