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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. Whichever is the two rings around the DNA. Interestingly, the majority of case, it seems clear that Ku must be removed from the contacts in the complex are with the sugar–phosphate DNA to allow DNA replication to proceed unhindered. backbone. A loop-like structure, extending between βP and βQ of each β barrel domain, interacts with the minor The relative accessibility of the DNA in the Ku–DNA groove of the DNA. However, no direct base contacts are complex also provides a nice explanation for how other R922 Current Biology Vol 11 No 22

Figure 2

(a) (b) Top View Direction of Ku translocation Ku80 VWA SAP domain β-bridge

α-helix 15 DNA α-helix 15

VWA VWA β-cradle

β−Bridge Ku80 Ku70

DNA End Ku70 Carboxy-terminal Carboxy-terminal DNA-PK VWA arm domain arm domain α-helix 14 domain β β P- Q β-barrel loops domains Side View

Current Biology

Structure of the Ku heterodimer–DNA complex. (a) A side view of the highly accessible to the solvent, possibly allowing other proteins, such Ku–DNA complex, looking down the helix axis of the DNA. Ku70 is as DNA ligase IV and certain nucleases, to bind directly. This view also coloured in shades of blue, Ku80 in pink and the double-stranded DNA highlights the intrinsic asymmetry within the Ku heterodimer complex. in grey. (b) A top view of the Ku–DNA complex shows two β-strands Although the extreme carboxy-terminal SAP (Ku70) and DNA-PKcs (βj) forming a narrow β-bridge structure that extends across the DNA binding domains (Ku80) are not present in the structure, their relative (represented as a space-filling model in grey). The top of the DNA is positions are indicated.

proteins might be able to gain access to the DNA ends to amino-terminal VWA domain, seems to buttress the facilitate their ligation (Figure 2b). For example, the pillars of the arch, acting as a structural support (Figure structure helps to explain how Ku can interact with and 2a). Indeed, the structural B-factors indicate that this potentiate the end-joining capacity of DNA ligase IV region of the structure is extremely stable. Could cleavage in vitro [6]. Mammalian DNA-PKcs also interacts with the or disruption of the α15 helix destabilise the complex, Ku–DNA complex, but the structure does not immedi- allowing the Ku to dissociate from the DNA? Interest- ately suggest how this might take place because Ku80 has ingly, point mutations in helix α14 of Ku80 completely been truncated in the crystal structure, presumably to abrogate its interaction with Ku70 [19]. This α-helix allow crystallisation, and is devoid of the DNA-PKcs inter- packs between helices α12 and α16, and disruption of action domain at its extreme carboxyl terminus [10]. In local interactions may ultimately destabilise the carboxy- regard to the accessibility of the DNA in the Ku–DNA terminal arm region, suggesting that this region of Ku complex, it is also noteworthy that most naturally occur- plays an important role in heterodimerisation. This is sup- ring DNA double-strand breaks require limited nuclease ported by reports that the carboxy-terminal arm regions of action before they can be ligated to another DNA end. Ku70 and Ku80 are sufficent to mediate Ku heterodimeri- Nuclease enzymes, such as the MRE11–RAD50–NBS1 sation [10,12–14,19]. complex, may play a role in these events [18], and the structure of the Ku–DNA complex might be designed to In the DNA-free Ku structure, a tri-helical SAP domain allow such proteins to act (Figure 2a). is present at the carboxy-terminal end of the Ku70 subunit [9]. This observation is supported by the solution structure After the β-core, the polypeptide chain of each Ku of the same region of human Ku70, which was reported subunit extends across the face of the complex to form a recently [20]. The Ku70 SAP domain is most structurally mainly helical carboxy-terminal arm region (Figure 2a) similar to the DNA-binding domain of T4 endonucle- [9]. Helix α15 of this region, which is wedged between ase VII and the RNA-binding domain of the bacterial the pillars of the DNA-binding arch structure and the transcriptional termination factor Rho [20–22]. In the crystal Dispatch R923

structure, the SAP domain is positioned against the base of prokaryotic Ku proteins and the solution of the strucures the Ku80 VWA domain, with the predicted DNA recogni- of higher-order complexes containing mammalian Ku and tion helix exposed to the solvent [9]. Previous reports indi- other proteins are clearly major goals for future research. It cate that the SAP domain of Ku70 can directly bind to seems likely that such work will provide further exciting DNA, and Goldberg and colleagues [9] propose that it may insights into the roles that the Ku protein plays in NHEJ prevent the Ku heterodimer from moving inwards away and in other biological events. from the DNA end or could cause pausing of Ku at spe- cific internal DNA sequences. Acknowledgements We thank Jane Bradbury for valuable editorial and scientific assistance and L. Serpell for valuable assistance with the preparation of Figure 2. We apol- Alternatively, it may be that the SAP domain provides Ku ogise to those whose work was not cited due to manuscript space con- with an entirely distinct mode of DNA binding. In this straints. Work in the SPJ laboratory is supported by grants from the Cancer regard, it is noteworthy that the Ku70 SAP domain is Research Campaign, the Association for International Cancer Research and the A-T Medical Research Trust. AJD is a Royal Society University Research distantly related to the DNA-binding domain of Myb, and Fellow and work in the AJD laboratory is supported by grants from Cancer that Myb domains are frequently present in proteins Research Campaign, the Association for International Cancer Research, the involved in telomere maintenance [23]. Furthermore, Royal Society and the Leukaemia Research Fund. disruption of this domain in yeast Ku70 does not affect DNA binding or NHEJ, but does lead to shortened telomeres [24], References 1. 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Wu X, Lieber MR: Protein-protein and protein-DNA interaction The remarkable similarity between the eukaryotic and regions within the DNA end-binding protein Ku70-Ku86. Mol Cell bacterial Ku homologues suggests that they derive from a Biol 1996, 16:5186-5193. common ancestral gene, the product of which presumably 13. Wang J, Dong X, Myung K, Hendrickson EA, Reeves WH: Identification of two domains of the p70 Ku protein mediating functioned as a homodimeric complex. Intriguingly, the dimerization with p80 and DNA binding. J Biol Chem 1998, genomes of several bacteria, including the symbiotic 273:842-848. 14. Osipovich O, Duhe RJ, Hasty P, Durum SK, Muegge K: bacteria Mesorhizobium loti and Sinorhizobium loti and also Defining functional domains of Ku80: DNA end binding and Agrobacterium tumefaciens, contain two distinct bacterial Ku survival after radiation. Biochem Biophys Res Commun 1999, genes in the same operon [21]. Could these encode 261:802-807. 15. Li B, Comai L: Requirements for the nucleolytic processing of DNA components of primitive heterodimeric Ku complexes? ends by the Werner syndrome protein-Ku70/80 complex. Presumably, the eukaryotic Ku proteins have evolved and J Biol Chem 2001, 276:9896-9902. acquired additional domains, such as the VWA, SAP and 16. Yoo S, Kimzey A, Dynan WS: Photocross-linking of an oriented DNA repair complex - Ku bound at a single DNA end. J Biol Chem DNA-PKcs interaction domains, to enhance the repair 1999, 274:20034-20039. activities of the complex or to provide additional functions, 17. Bliss TM, Lane DP: Ku selectively transfers between DNA molecules with homologous ends. J Biol Chem 1997, such as roles in telomere maintenance or in the control of 272:5765-5773. chromatin structure. Examination of the structures of the 18. Haber JE: The many interfaces of Mre11. Cell 1997, 95:583-586. R924 Current Biology Vol 11 No 22

19. Osipovich O, Durum SK, Muegge K: Defining the minimal domain of Ku80 for interaction with Ku70. J Biol Chem 1997, 272:27259-27265. 20. Zhang Z, Zhu L, Lin D, Chen F, Chen DJ, Chen Y: The three- dimensional structure of the C-terminal DNA-binding domain of human Ku70. J Biol Chem 2001, in press. 21. Doherty AJ, Jackson SP,Weller GR: Identification of bacterial homologues of the Ku DNA repair proteins. FEBS Lett 2001, 500:186-188. 22. Aravind L, Koonin EV: Prokaryotic homologs of the eukaryotic DNA-end-binding protein ku, novel domains in the ku protein andprediction of a prokaryotic double-strand break repair system. Genome Res 2001, 11:1365-1374. 23. Bilaud T, Brun C, Ancelin K, Koering CE, Laroche T, Gilson E: Telomeric localization of TRF2, a novel human telobox protein. Nat Genet 1997, 17:236-239. 24. Diller L, Wellinger RJ, Larrivee M, Kremmer E, Jaklin S, Feldmann HM: A short C-terminal domain of Yku70p is essential for telomere maintenance. J Biol Chem 2000, 275:24921-24927.