Joint Sino–U.K. Protein Symposium: a Meeting to Celebrate the Centenary of the Biochemical Society 1387 Non-homologous end-joining partners in a helical dance: structural studies of XLF–XRCC4 interactions Qian Wu* 1 , Takashi Ochi*, Dijana Matak-Vinkovic†, Carol V. Robinson‡, Dimitri Y. Chirgadze* and Tom L. Blundell* *Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, U.K., †University Chemical Laboratory, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K., and ‡Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, U.K. Abstract XRCC4 (X-ray cross-complementation group 4) and XLF (XRCC4-like factor) are two essential interacting proteins in the human NHEJ (non-homologous end-joining) pathway that repairs DNA DSBs (double- strand breaks). The individual crystal structures show that the dimeric proteins are homologues with protomers containing head domains and helical coiled-coil tails related by approximate two-fold symmetry. Biochemical, mutagenesis, biophysical and structural studies have identified the regions of interaction between the two proteins and suggested models for the XLF–XRCC4 complex. An 8.5 Å (1 Å = 0.1 nm) resolution crystal structure of XLF–XRCC4 solved by molecular replacement, together with gel filtration and nano-ESI (nano-electrospray ionization)–MS results, demonstrates that XLF and XRCC4 dimers interact through their head domains and form an alternating left-handed helical structure with polypeptide coiled coils and pseudo-dyads of individual XLF and XRCC4 dimers at right angles to the helical axis. XLF and XRCC4 play roles in recruiting and stabilizing DNA ligase IV at DSBs in NHEJ DNA DSBs (double-strand breaks) can be caused by ionizing radiation or toxic chemical exposure, but are also present as intermediates in V(D)J recombination and class switch recombination for antigen receptor diversity formation. Unrepaired DSBs lead to chromosome fragmentation and rearrangement and are lethal to cells, changing cell gene regulation and expression, and often leading to cancer cell formation. The two major DSB repair pathways are HR (homologous recombination) and NHEJ (non-homologous end-joining). Our current understanding of the NHEJ repair pathway (Figure 1) is that it comprises three major steps: first, the Ku heterodimer and DNA-PKcs (DNA-dependent protein kinase catalytic subunit) recognize DSBs and generate a protein-binding platform for XRCC4 (X-ray cross-complementation group 4), XLF (XRCC4-like factor) and other proteins [1,2]; secondly, Artemis containing endonuclease activity and other end-processing proteins, such as PNKP (polynucleotide kinase/phosphatase) and PolX family DNA polymerases, process the DSBs ends before ligation [3,4]; and thirdly, the XRCC4–LigIV (DNA ligase IV) complex ligates the two ends of the DNA promoted by XLF [5]. Understanding how these transient NHEJ Key words: double-strand break (DSB), non-homologous end-joining (NHEJ), X-ray cross- complementation group 4 (XRCC4), XRCC4-like factor (XLF). Abbreviations used: ATM, ataxia telangiectasia mutated; BRCT, BRCA1 C-terminal; DNA- PKcs, DNA-dependent protein kinase catalytic subunit; DSB, double-strand break; EM, electron microscopy; LigIV, DNA ligase IV; nano-ESI, nano-electrospray ionization; NHEJ, non-homologous end-joining; PNKP, polynucleotide kinase/phosphatase; SAXS, small-angle X-ray scattering; XRCC4, X-ray cross-complementation group 4; XLF, XRCC4-like factor. 1 To whom correspondence should be addressed (email qw222@cam.ac.uk). complexes assemble structurally in both space and time is a challenging, but timely, research focus. In the past, we have defined the crystal structures of XRCC4 with LigIV peptide [6], XLF [7] and more recently DNA-PKcs [8]. The next step in exploring the NHEJ protein assembly is to study the complexes of these key protein components. Although XLF itself cannot directly ligate DSBs, it performs an essential NHEJ function by interacting with XRCC4 and stabilizing XRCC4–LigIV broken DNA ends, thereby enhancing the LigIV end-joining process [9]. The mechanism of XLF mediating ligation enhancement is through enhancement of LigIV recharging following ligation in the presence of ATP [10]. How XLF is structurally involved in the NHEJ pathway is not clear. In the present paper, we focus on current biophysical studies of XLF and XRCC4 and XLF–XRCC4 interactions and recent results from the crystal structure of the XLF–XRCC4 complex, which shed light on this question. XLF and XRCC4 are dimeric coiled-coil proteins with a common ancestor Despite the low sequence identity (13.6%), the crystal structures of XLF and XRCC4 demonstrate that the two proteins are homologous homodimers comprising globular head domains and C-terminal helices that form coiled-coil tail structures [6,7,12,13]. However, the structural differences between the two are large. The head domains form seven- stranded antiparallel β -sheets sandwiching a helix–turn–helix motif between β 4 and β 5, but XLF contains an extra helix in the N-terminal region. Whereas the tail structure of XRCC4 comprises an elongated coiled-coil, the equivalent extended helix α4 of XLF is followed by further helices, α5 and α6, Biochem. Soc. Trans. (2011) 39, 1387–1392; doi:10.1042/BST0391387 C  The Authors Journal compilation C  2011 Biochemical Society