View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector R920 Dispatch DNA repair: How Ku makes ends meet Aidan J. Doherty* and Stephen P. Jackson† The recently determined crystal structure of the Ku of NHEJ, structural and functional homologues of Ku70, heterodimer, in both DNA-bound and unbound forms, Ku80, ligase IV and XRCC4 exist in lower eukaryotes, has shed new light on the mechanism by which this including yeast. DNA-PKcs, however, seems to be restricted protein fulfills its key role in the repair of DNA to vertebrates. double-strand breaks. Biochemical studies have yielded some major insights into Address: *Cambridge Institute for Medical Research, Department of Haematology, University of Cambridge, Hills Road, the mechanisms of NHEJ. Most notably perhaps, the Cambridge CB2 2XY, UK. †Wellcome Trust and Cancer Research observation that Ku binds tightly and specifically to a Campaign Institute of Cancer and Developmental Biology and variety of DNA end-structures, including blunt ends, 5′ or Department of Zoology, University of Cambridge, Tennis Court Road, 3′ overhangs and DNA hairpins, suggests that it acts as the Cambridge CB2 1QR, UK. primary sensor of broken chromosomal DNA [3,5]. Once E-mail: *[email protected]; †[email protected] bound to a DNA double-strand break, Ku presumably Current Biology 2001, 11:R920–R924 then recruits DNA-PKcs, whose protein kinase function — which in vitro preferentially targets proteins bound in 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. cis on the same DNA molecule — could trigger changes in chromatin structure at the site of the DNA double-strand DNA double-strand breaks are generated by ionising breaks and/or regulate the activities of other repair factors radiation and radio-mimetic chemicals. They also occur (Figure 1). during V(D)J recombination, which occurs in developing mammalian lymphocytes to help generate the antigen- In addition, the Ku–DNA-PKcs complex might physically binding diversity of immunoglobulin and T-cell receptor recruit other repair factors. In line with this idea, Ku proteins. It is of crucial importance that cells recognise interacts biochemically with the ligase IV–XRCC4 double-strand breaks and effect their efficient repair, complex [6] and is required for the analogous complex in because a single unrepaired cellular double-strand break the budding yeast Saccharomyces cerevisiae to localise to can be sufficient to induce cell death, and defective DNA double-strand breaks in vivo [7]. Other potential double-strand break repair can lead to mutations or the NHEJ functions for Ku include protecting DNA ends loss of large tracts of chromosomal material. In eukaryotic from excessive nucleolytic attack, helping to align two cells, double-strand breaks are generally repaired either broken DNA termini, and targeting DNA double-strand by a homologous recombination pathway involving the breaks to appropriate sites of repair within the nucleus. In Rad51 and Rad52 proteins, or via DNA non-homologous addition to its roles in DNA repair, Ku also functions in end joining (NHEJ) [1]. Both pathways are present in the maintenance of telomeres — the protective caps at the all eukaryotes studied but their relative use differs ends of linear chromosomes. Indeed, loss of Ku leads to between species. telomeric shortening and to enhanced rates of chromo- some end-fusions (see for example [8]). The NHEJ pathway in mammals requires the DNA- dependent protein kinase (DNA-PK) — a multi-subunit A significant landmark in our understanding of the NHEJ serine/threonine kinase consisting of an approximately apparatus has now been reported by Goldberg and col- 470 kDa catalytic subunit, DNA-PKcs, and a DNA end- leagues [9], who have elucidated the crystal structure of binding component called Ku [2,3]. Ku is itself a het- the Ku heterodimer, both alone and bound to a cleverly erodimer of two proteins termed Ku70 (69 kDa) and Ku80 designed DNA substrate with a single accessible end (83 kDa). Cells or animals deficient in these proteins are (Figure 2). Consistent with the previously reported defective in double-strand break rejoining and are hyper- sequence homologies between Ku70 and Ku80, which sensitive to ionising radiation and various radio-mimetic indicated that Ku70 and Ku80 are derived from a common agents [1–3]. Furthermore, because of their inability to cor- ancestor [5,10], the overall folds of the Ku70 and Ku80 rectly rejoin V(D)J recombination intermediates, animals subunits are remarkably similar and the two proteins form lacking Ku or DNA-PKcs display severe-combined a quasi-symmetrical structure. Overall, Ku70 and Ku80 immunodeficiency. Other NHEJ components in mammals have a common three-domain architecture composed of an are the DNA ligase IV–XRCC4 complex [1–3] and the amino-terminal α/β domain, an unusual β-barrel domain recently described Artemis protein, a deficit in which leads and a helical carboxy-terminal arm structure (Figure 2). to radiosensitivity and severe-combined immunodeficiency The arrangement of the proteins is such that the complex in humans [4]. In line with the evolutionary conservation has an open ring-like shape with the DNA threaded Dispatch R921 through the aperture. This immediately explains why Ku Figure 1 binds to DNA ends rather than closed circular DNA, and provides clues to how it operates in NHEJ. Rad50 Mre11 The amino-terminal region of each Ku subunit, located on NBS1 LigaseLigase IV IV BRCTBRCT XRCC4 the exterior of the complex, comprises a compact α/β XRCC4 domain with a six-stranded β-sheet of the Rossman fold type (Figure 2). This domain is a divergent member of the Ku80 von Willebrand factor A (VWA) family of domains [11]. Ku70 Although these were first identified in extracellular DNA-PKcs eukaryotic proteins that mediate cell adhesion, they have been subsequently identified on intracellular proteins and in prokaryotes [11]. As now explained by the Ku crystal Recruitment/regulation of Additional roles: protecting DNA structure [9], a number of groups have found previously DNA repair factors. from nucleases; aligning DNA that deletion of the amino-terminal regions of Ku70 or Effects on replication, ends; targeting DSBs to repair Ku80 does not affect heterodimerisation or DNA end transcription and chromatin? sites; telomere maintenance binding (see for example [12–14]). Current Biology Instead, and consistent with the functions of other The involvement of Ku in NHEJ. After binding to a DNA double-strand characterised VWA domains, the available data suggest a break within the cell, Ku is thought to recruit DNA-PKcs. This protein role for these domains of Ku in mediating its contacts with kinase then recruits and regulates a number of other factors involved in other proteins. For example, the Werner’s syndrome double-strand break repair. In addition, Ku itself recruits two complexes protein, WRN, which has been linked to DNA-repair involved in DNA damage signalling and repair: the Mre11–Rad50–NBS1 and ligase IV–XRCC4 complexes. Other roles events, has been shown to bind to the amino-terminal for Ku in double-strand break (DSB) repair and general maintenance of region of Ku80 that contains the VWA domain [15]. It is genome integrity are less well-defined. tempting to speculate that the VWA domains of Ku sequester WRN and other proteins at sites of DNA damage, forming larger protein complexes that are required for con- made here or elsewhere in the complex, providing an certed DNA repair. Another attractive possibility is that explanation for why Ku lacks significant sequence prefer- associations in trans between the VWA domains of two ence for DNA. The combination of the extended β-barrel DNA-bound Ku heterodimers provide a mechanism to cradle and a narrow β-bridge allows Ku to interact with two bring together and structurally align two broken DNA turns of the DNA double helix while still exposing much ends, ready for their religation. of the DNA to the solvent (Figure 2b). The central seven-stranded anti-parallel β-barrel domains This unusual mode of DNA binding by Ku explains of Ku70 and Ku80 form the cradle of the DNA-binding previous biochemical studies revealing that, once bound to toroid (Figure 2). A large insertion in the centre of the a DNA end, Ku can translocate internally along the DNA β-barrel, between strands βG and βN, forms an unusual fragment and can be transferred from one linear DNA bridge-like structure, the β-bridge, through which the DNA molecule to another if the ends of the two DNA molecules is threaded. The preformed channel fits exquisitely around can base-pair with one another [17]. But a major question DNA in a preferred asymmetric orientation, similar to raised by the structure is that of how the threaded Ku threading a nut onto a bolt. This allows Ku to move complex becomes dissociated from the DNA once the inwards away from the DNA end with Ku70 located proxi- DNA has religated? In solution, the Ku heterodimer is mal and Ku80 distal to the end, as shown previously by very stable, and the crystal structures of Ku in its DNA- biochemical analyses [16]. The inner surface of the ring bound and DNA-free forms are remarkably similar. One and the β-barrel cradle are lined with positively charged possibility, therefore, is that the heterodimer dissociates residues, complementing the negative charge of the DNA from the DNA before or concomitant with the DNA liga- and stabilising DNA end binding. tion process. Alternatively, Ku’s removal from the DNA might require either a major structural rearrangement of As shown in Figure 2a, the Ku complex effectively wraps the protein or its proteolytic cleavage.