DNA Damage Recognition, Signalling and Repair (Cancer Research UK DNA Repair Enzymes Group)

Pearl Group

Section: Section of Structural Biology

DNA Double Strand Break Repair

(JA Hinks), (J Maman), L Spagnolo, LH Pearl; in collaboration with O Llorca, CIB-CSIC Madrid, P Driscoll, UCL

Source of external funding: Cancer Research UK

Ionising radiation can cause several types of DNA lesions, one of which is the double stand break (DSB). Unrepaired or improperly repaired DSBs can cause cell death, cell cycle arrest and chromosome translocations, resulting in increased rates of mutation and ultimately in carcinogenesis. The major pathway for repair of Dsbs in mammalian cells is non-homologous end joining (NHEJ) system. Repair in this system is initiated by the heterodimeric DNA end-binding protein Ku70:86, which slides over free DNA ends and helps recruit the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) which binds and is activated by broken DNA ends. The DNA-bound Ku - DNA-PK complex promotes association of free-DNA ends and recruits a second complex XRCC4 - Lig IV that ligates the blunt ends. A specialised endonuclease, Artemis, also binds DNA-PK and is required for incision of DNA loops at broken ends, and production of ligatable blunt-ends.

We are using biochemical and structural techniques to understand the process of assembly and regulation of the NHEJ repair complex at DNA ends. In particular we wish to understand how broken DNA ends are recognised by DNA-PKcs, how this activates its inherent protein kinase activity, and how phosphorylation of other NHEJ components by DNA-PKcs affects their mutual interactions and activity. We also wish to understand the particular role of Artemis in processing DNA ends and how this activity is modulated by its direct association with DNA-PKcs. We have previously defined a C-terminal domain in Ku86, which is responsible for recruitment of DNA-PKcs, and established high-level recombinant expression of this, and have now determined its structure by NMR spectroscopy. We have also determined a low resolution structure of the 450kDa DNA-PKcs using single-particle electron microscopy, in free and DNA–bound conformations. These studies have revealed a mechanism of clamp-closure by DNA-PKcs on DNA binding, which correlates with its catalytic activation. In ongoing studies we are improving the resolution of our DNA-PKcs studies using electron cryo-microscopy, and extending these to complexes of DNA-PKcs, DNA and other NHEJ repair proteins. We are currently developing expression systems for Artemis that will allow its high-level expression in a soluble form for detailed structural studies.

DNA Single-Strand Gap Repair

AW Oliver, (S Jones), LH Pearl; in collaboration with GM de Murcia, École Supérieure de Biotechnologie de Strasbourg, France

Source of external funding: Cancer Research UK

Single-particle negative-stain electron micrograph of DNA-PKcs in complex with a DNA molecule

Breaks and small gaps in the continuity of a DNA strand result from damage by reactive oxygen species (exogenous or endogenous) or ionising radiation, as intermediates in the base-excision repair (BER) of damaged or inappropriate bases, as by products of aborted DNA Topo I activity, and by simple hydrolytic damage under physiological conditions. Although double-strand breaks (DSBs) are the most cytotoxic form of damage sustained by DNA, single-strand breaks and gaps (SSBs) occur with far higher frequency, and it is only the efficiency of the repair systems for SSBs that renders them apparently less dangerous. In fact, SSBs not repaired prior to DNA replication will be turned into DSBs once the complementary strand is displaced at the replication fork. SSBs are repaired via a dynamic multiprotein complex, recruited to the site of damage by the primary sensors PARP-1 and PARP-2, and coordinated by the XRCC1 scaffold protein. XRCC1 provides binding sites for PARPs, the dual-function polynucleotide 5'-kinase-3'phosphatase (PNK) which establishes the correct phosphorylation pattern in the gap, DNA polb which appends missing bases to the free 3' OH, and DNA ligase IIIa which closes the phosphodiester backbone.

We are using structural and biochemical techniques to define the mechanism of gap recognition by the primary sensors, and the recruitment and assembly of the other enzymatic components to the site of damage. We have previously crystallised the catalytic domain of the primary sensor PARP-2 and have now determined the crystal structure. Recombinant expression systems for production of all the components of the system have now been established and milligram quantities of protein are being produced for structural studies.