Discovery of DNA processing proteins that control genome stability [webcast of lecture]
The chemical reactivity of DNA contributes to the staggering array of DNA lesions that occur in cellular genomes every day. In addition to their potential mutagenicity, these DNA lesions can block important processes such as DNA replication, which can potentially prevent cell proliferation. It is vital that DNA damage is repaired rapidly to prevent mutations, rearrangements or changes in chromosome number from occurring. Cells have evolved sophisticated pathways that repair DNA damage, and stalled or broken DNA replication forks. Defects in these pathways can have serious consequences that range from cell death and embryonic lethality to a range of debilitating disease syndromes.
Nucleases play important roles DNA repair. In yeast for example, the structure–specific nuclease Rad1-Rad10 is required to trim excess DNA (DNA flaps) at the final stages of termed homologous recombination (HR), an ancient mechanism for repairing DNA damage and stalled or broken replication forks.In 2005 we discovered a regulatory subunit of Rad1-Rad10, termed Slx4, that acts as a scaffold not just for Rad1-Rad10 but for also for Slx1, a structure-specific nuclease of unknown function. In cells lacking Slx4, Rad1-Rad10 cannot cleave DNA flaps during HR-mediated repair of double-strand breaks, and so cells die. Slx4 appears to facilitate Rad1-Rad10 by deforming DNA flaps to render them accessible to attack. In 2009, my lab reported the identification of human SLX1, and SLX4 which binds to and regulates not only SLX1 but two other structure–specific repair nucleases: XPF–ERCC1 (human Rad1-Rad10) and MUS81–EME1 (which is closely related to XPF–ERCC1). We refer to the SLX4 complex is a “molecular toolkit” for DNA repair because the three nucleases can cleave a variety of branched DNA species that resemble HR intermediates. Cells depleted of SLX4 are particularly sensitive to agents that cause inter-strand cross-links (ICLs), toxic lesions that block DNA replication forks. The SLX4 complex processes branched intermediates that occur during the HR step of ICL repair required to regenerate an intact replication fork. In collaboration with Johan de Winter (Erasmus Medical Centre, Rotterdam) we discovered that biallelic mutations in SLX4 cause Fanconi anaemia (see below), further underscoring the importance of the SLX4 complex for human health. Intriguingly, the SLX1 nuclease can cleave Holliday junctions (HJs), DNA structures that arise not only during DNA repair but also during meiosis. Recent data suggests that SLX1 is involved in HJ resolution in vivo in germ cells.
Fanconi anaemia (FA) is a rare inherited chromosome instability syndrome accompanied by developmental and skeletal defects, bone marrow failure and predisposition to cancer. There are fifteen FANC proteins, and the central component of the FA pathway is FANCD2, which is mono–ubiquitylated at Lys561 in S–phase and in response to ICLs. This is catalysed by the eight–subunit FA core complex. The mono-ubiquitylation of FANCD2 is essential for the repair of DNA inter-strand crosslinks (ICLs) but despite much work in this area exactly how mono–ubiquitylation of FANCD2 promotes ICL repair at the molecular level was unknown. In 2010, my lab made a major breakthrough by showing thJohn's medal was presented by Prof Andrew Calder, National Chairman of Tenovus Scotland and Prof Anna Dominiczak, Head of College, College of Medical, Veterinary and Life Sciencesat the UBZ domain of the previously uncharacterized FAN1 protein interacts with the mono-ubiquitylated form of FANCD2, and that FAN1 is recruited to sites of DNA damage in a manner that requires FANCD2 mono–ubiquitylation. FAN1 is a structure–specific nuclease that is specific for 5’ DNA flaps. Intriguingly, like the SLX4 complex, FAN1 appears to process DNA repair intermediates during the HR stage of ICL repair to enable regeneration of an intact replication fork. So binding of FAN1 to mono-ubiquitylated FANCD2 at least partly explains how FANCD2 mono–ubiquitylation regulates DNA repair. However, the available evidence indicates that there must be other ligands of mono-ubiquitylated FANCD2 that regulate ICL repair, and we recently identified several such ligands.
When replication forks encounter nicks in the DNA backbone, forks can collapse and when this happens there is a high risk of genome instability. Not only must the ends of the broken fork be captured and protected from further degradation, but an intact replication fork must be regenerated and this is achieved by HR. We found that budding yeast Mms22 is required for HR-mediated repair of stalled or broken DNA replication forks, and in 2010 we reported the identification of a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NFκBIL2/TONSL, of unknown function. Both MMS22L and TONSL bind in the vicinity of distressed replication forks and, by mechanisms that are not yet clear, they promote the loading of the RAD51 recombinase to enable HR–mediated fork repair. My lab is currently using a range of genetic models to understand the molecular modes of action of SLX4, FAN1, MMS22L–TONSL and other new regulators of genome stability we have identified. We are also starting to develop small molecule inhibitors of some of these proteins as next-generation anti-cancer drugs.
Muñoz, I. M., Hain, K., Déclais, A.–C., Gardiner, M., Toh, G. W., Sanchez-Pulido, L., Heuckmann, J., Toth, R., Macartney, T., Eppink, B., Kanaar, R., Ponting, C. P., Lilley, D. M. J. and Rouse, J. (2009) Coordination of structure–specific nucleases by human SLX4/BTBD12 is required for DNA repair. Mol. Cell 35, 116–127.
MacKay, C., Declais, A.–C., Lundin, C., Agostinho, A., Deans, A.J., MacArtney, T.J., Hofmann, K., Gartner, A., West, S.C., Helleday, T., Lilley, D.M.J, and Rouse, J. (2010) Identification of KIAA1018/FAN1, a DNA repair endonuclease recruited to DNA damage by mono-ubiquitinated FANCD2. Cell 142, 65-76.
Duro, E., Lundin, C., Ask, K., Sanchez–Pulido, Macartney, T.J., Toth, R., Ponting, C.P., Groth, A., Helleday and Rouse, J. (2010) Identification of the MMS22L–TONSL complex that promotes homologous recombination. Mol. Cell. 40, 632-644.
Stoepker, C., Hain, K., Schuster, B., Hilhorst–Hofstee Y., Rooimans, M.A., Steltenpool, J., Oostra, A.B., Eirich, K., Korthof, E.T., Nieuwint, A.W.M, Jaaspers, Bettecken, T., N.J.G., Joenje, H., Schindler, D.*, Rouse, J.* and de Winter, J. P.* (2011) SLX4, a coordinator of structure–specific endonucleases, is mutated in a new Fanconi anemia subtype. Nat. Genet. (2011) Jan. 16th Advance online publication.