View metadata, citation and similar papers at core.ac.uk brought to you by CORE R622 Dispatch provided by Elsevier - Publisher Connector

DNA repair: The Nijmegen breakage syndrome protein Carol Featherstone and Stephen P. Jackson

The gene mutated in Nijmegen breakage syndrome, a gene appears to be involved in signalling DNA damage to chromosome instability disorder, has been identified p53 [6]. Mapping of the gene affected in NBS — termed and sequenced. The protein product of this gene forms NBS1 — to a 1 centimorgan region on chromosome 8q21 a complex with hMre11 and hRad50 — proteins that are [7] and, by a method of chromosome transfer involving involved in repairing double-strand breaks in DNA. cell fusion, to chromosome 8q21–24 [8] strongly suggested that NBS1 was distinct from ATM. Address: Wellcome/Cancer Research Campaign Institute and Department of Zoology, Cambridge University, Tennis Court Road, Cambridge CB2 1QR, UK. This conclusion is now unequivocal since the chromoso- mal mapping location of the NBS1 gene was confirmed Current Biology 1998, 8:R622–R625 recently by positional cloning [9,10]. Nevertheless, the http://biomednet.com/elecref/09609822008R0622 phenotypic similarities between the two diseases, and the © Current Biology Publications ISSN 0960-9822 lack of complementation for radiation-induced chromoso- mal aberrations in NBS–AT cell fusions [11], suggest that Nijmegen breakage syndrome (NBS), an autosomal reces- the two gene products may work closely in the same bio- sive disease [1], is a relatively rare disorder with only about chemical pathway, and perhaps even in the same protein 70 affected families worldwide. The key clinical features complex [11]. In an unrelated study, Petrini and col- of NBS include stunted growth; microcephaly (abnormally leagues [12] identified the protein encoded by the NBS1 small head); respiratory infections due, most probably, to gene as a component of a complex that is implicated in the immunodeficiency caused by immune system defects; repairing the DNA double-strand breaks that are induced and predisposition to cancer at an early age, particularly to by DNA-damaging agents and that occur naturally during leukaemia, lymphoma, haemoblastoma or neuroblastoma. meiotic recombination. These findings provide a frame- Some striking clinical and, especially, cellular similarities work within which the molecular basis of NBS might be to the autosomal recessive disease ataxia telangiectasia explained as a defect in signalling and/or repairing DNA (AT) have led NBS to be classified as a variant of AT; double-strand breaks. however, NBS patients have neither ataxia (lack of muscu- lar coordination) nor telangiectasia (abnormal vasodila- The NBS1 protein tion), and AT patients do not have microcephaly. The NBS1 gene encodes a polypeptide of 754 amino acids with a predicted molecular mass of 85 kDa that Varon et The similarities in the characteristics of cells from NBS al. [9] named nibrin, and Petrini and colleagues [12] call and AT patients nonetheless suggest that the defective NBS1. Most cases of NBS are due to a common founder functions in both diseases might be related. In particular, mutation in NBS1. The mutation, 657del5, is a 5 base pair both NBS and AT cells display chromosomal instability, deletion that results in premature truncation of the most notably of chromosomes 7 and 14 [2,3] where the protein product 15 amino acids downstream of the dele- genes for immunoglobulins and T-cell receptors are tion site [9,10]. Overall, NBS1 has no striking primary located. Also, in both conditions the cells are hypersensi- sequence similarity to previously characterized proteins. tive to ionizing radiation, their chromosomes have high It has two recognizable domains, however, within the levels of radiation-induced chromosome fragility, and amino-terminal 200 amino acids: one forkhead-associated DNA replication is not delayed in response to ionizing (FHA) domain and one breast cancer carboxy-terminal radiation like it is in normal cells [2]. This cell-cycle (BRCT) domain (Figure 1). Both types of domain have checkpoint defect may be due, in part, to a delayed and been found individually in a variety of proteins but they reduced induction of the checkpoint tumour suppressor have never before been found adjacent to each other protein p53 in response to irradiation [4,5]. within the same protein.

The defective gene in AT, ATM, maps to chromosome The BRCT domain was first identified in the carboxyl ter- 11q23 and encodes a member of the phosphatidylinositol- minus of the breast cancer susceptibility protein BRCA1 3 kinase family that is related to the catalytic subunit of [13] and, to date, has been found in over 40 other proteins DNA-dependent protein kinase (DNA–PKcs) — a protein that function predominantly in DNA repair and DNA involved in DNA double-strand-break repair and in damage checkpoints [14]. On structural grounds, the BRCT recombination of the V(D)J regions of immunoglobulin domain is expected to be involved in protein–protein inter- and T-cell receptor genes, which generates antibody and actions, and this seems to be the case for the domains in antigen receptor diversity [6]. The product of the ATM DNA ligase III [15] and poly(ADP-ribose) polymerase [16] Dispatch R623

Figure 1

The domain structure of NBS1 is shown diagrammatically. It comprises adjacent FHA GR H and BRCT domains spanning the amino- terminal 200 amino acids of the polypeptide 55–75 and a 550 amino acid tail with no apparent homology to other proteins. In general, FHA 657del5 domains are 55–75 amino acids in size with indistinct boundaries. They contain three N FHA BRCT C highly conserved blocks (blue) that are 750 predicted to form β strands, separated by more divergent spacer sequences. The only invariant residues are a glycine–arginine pair (GR) in the first block and a histidine (H) in ~95 the second. The FHA domain is distinct from the forkhead domain (FD) that is also found in G/A W the forkhead family of transcription factors. ABCDE Current Biology BRCT domains are about 95 residues long and comprise five highly conserved blocks (yellow) designated A–E. The only invariant frequently G or A. The domain is predicted to arrow indicates the location of the common residue is a tryptophan (W) in block D. The contain four β strands, probably forming a founder mutation in NBS patients (657del5). two residues at the end of block B are core sheet structure, and two α helices. The

that bind to XRCC1, and the domain in DNA ligase IV that formed by ionizing radiation, and in the initiation and pro- binds to XRCC4 [17]. cessing of DNA double-strand breaks in meiotic recombi- nation [21]. Rad50p and Mre11p appear to be homologues The FHA domain was originally identified in a subset of of the Escherichia coli proteins SbcC and SbcD, respec- the forkhead family of transcription factors, and has subse- tively [22], which together have DNA exonuclease and quently been found in around 20 unrelated proteins [18]. endonuclease activity [23], suggesting that Mre11p and Like NBS1, all but one of the characterized FHA-contain- Rad50p might also have these activities. ing proteins are nuclear, and most of the uncharacterized proteins contain putative bipartite nuclear localization Carney et al. [12] isolated a complex from human cells that signals. Four of the FHA-domain-containing proteins are contained hMre11, hRad50 and several other polypep- yeast protein kinases that are involved in meiotic recombi- tides. They obtained sequence information for a copurify- nation and respond to signals related to DNA replication ing polypeptide of 95 kDa — p95 — and used it to and repair. As yet, there is no known function for FHA construct a cDNA from overlapping expressed sequence domains. There is a hint, however, that the domains might tags in the human genome database and to predict the mediate interactions with phosphoproteins: the FHA- amino acid sequence of p95. The gene encoding p95 was domain-containing kinase-associated protein phosphatase localized to chromosome 8q21.3 indicating that it was (KAPP) from Arabidopsis thaliana interacts with its target probably the gene defective in NBS patients that had kinase only when the kinase is autophosphorylated on been mapped to this location the previous year. The serine and threonine residues, and the site of this interac- recent sequencing of the NBS1 gene [9,10] verified that tion maps to the region in KAPP that contains the FHA NBS1 was identical to the gene encoding p95 [12]. domain [19]. By analogy, NBS1 might therefore interact Further confirmation was provided by the finding that the with a phosphoprotein in the DNA repair apparatus. p95 protein and its mRNA are absent from NBS cells [12].

The function of NBS1 Carney and colleagues found that NBS1 binds to hMre11 NBS1 was found to have a role in DNA repair virtually at in a yeast two-hybrid protein–protein interaction screen. the same time as the cloning of the NBS1 gene when Together with hMre11, hRad50 and possibly other Carney et al. [12] discovered that the gene encoding p95, a polypeptides, NBS1 forms a complex with an estimated component of the human Mre11–Rad50-containing DNA molecular mass of 1600 kDa [12]. NBS1 is not required for repair complex, was identical to NBS1. The MRE11 and the interaction between hMre11 and hRad50, but it does RAD50 genes were originally identified from mutations in seem to be required for the subcellular relocation of these the budding yeast Saccharomyces cerevisiae that cause proteins in response to DNA damage. Before treatment of hypersensitivity to ionizing radiation and an inability to cells with ionizing radiation, both hMre11 and hRad50 are initiate and/or complete meiosis [20]. Both gene products distributed diffusely throughout the nucleoplasm, pre- are involved in repairing DNA double-strand breaks sumably in a complex. After irradiation, however, hMre11 R624 Current Biology, Vol 8 No 17

and hRad50 are found in foci within the nucleus [24]. Breaks that form in response to DNA-damaging agents, These foci may be individual sites of double-strand breaks such as ionizing radiation, might be recognized directly by in the DNA or they may correspond to subnuclear struc- NBS1 or, more likely, NBS1 might recognize a signal from tures that somehow organize the DNA repair machinery. a DNA-break-detecting complex, which may contain By contrast, these foci of hMre11 and hRad50 do not DNA–PK and/or ATM (Figure 2). By analogy with the appear in response to ionizing radiation in cells that lack Arabidopsis protein KAPP, this signal might be phosphory- NBS1. Formation of the foci is also drastically reduced in lation on a serine or threonine residue in a target protein AT cells [24], suggesting that ATM might recruit the that would then be recognized by the FHA domain in hMre11–hRad50 complex to the double-strand break or NBS1. Alternatively, other protein–protein interactions trigger signalling pathways that result in the relocation of might be involved, for example through the BRCT the complex. domain in NBS1. In binding to a component at the double-strand break, NBS1 might act as an adaptor bring- In S. cerevisiae, Mre11p and Rad50p form a complex with ing the Mre11–Rad50 exonuclease activity to the DNA at least one other protein (of approximately 95 kDa), ends to prepare them for ligation. In doing so, it may also Xrs2p, which is also involved in repairing double-strand recruit other components in the complex to facilitate the DNA breaks [20]. In the human complex, the p95 protein repair process. Because NBS cells are defective in the — which is now known to be NBS1 — was interesting at control of cell-cycle checkpoints [4,5], NBS1 itself, or the first because of its similarity in size to Xrs2p, indicating consequences of its action, must also impinge on the that it might be the human homologue of this protein. DNA-damage signalling pathways to induce a delay in Surprisingly, the sequences of the two proteins are rela- DNA replication. tively dissimilar, having a sequence identity of 28% only over their first 115 amino acid residues. No other protein Towards a molecular explanation of NBS encoded in the yeast genome appears to be a closer rela- This new knowledge of the NBS1 gene and the role of its tive of NBS1, however, so NBS1 might be a functional product in the complex containing hMre11 and hRad50 analogue or a distantly related homologue of Xrs2p. suggest how the cellular phenotypes of NBS cells might arise. Certain features of NBS, including chromosomal A model for the function of the NBS1–Mre11–Rad50 instability, sensitivity to DNA-damaging agents and failure complex to induce checkpoint controls, might be explained by the How then might NBS1 function? From its association with absence of a protein that is crucial for recognizing, sig- hMre11 and hRad50, and its similarity with Xrs2p, it nalling and repairing DNA double-strand breaks. The clin- seems likely that NBS1 will function in the recognition, ical symptoms of immunodeficiency might be explained if repair and/or signalling of DNA double-strand breaks. there are defects in the essential gene rearrangements that

Figure 2

A model for the function of the NBS1 protein. Ionizing NBS1 is part of a ~1600 kDa complex that radiation contains hMre11, hRad50 and, most likely, other proteins. When double-strand breaks occur in the DNA, for example upon treatment with ionizing radiation or with radiomimetic Cytoplasm Nucleus drugs such as bleomycin, they are most probably recognized by a complex of proteins that may include, or interact with, ATM or

DNA-damage- DNA–PK. One of these kinases may then recognition phosphorylate a substrate in the complex that complex provides a recognition site for the NBS1 ATM NBS1–hMre11–hRad50 complex, and/or some other type of protein–protein interaction

hRad50 may occur, causing the relocalization of the hMre11 complex from diffuse points in the nucleus to foci of DNA repair. The recruited NBS1–hMre11–hRad50 complex may then p53 promote repair, for example by exonuclease Degradation action on the DNA ends, and may also signal to the cell-cycle checkpoint machinery, for example to prevent p53 turnover thereby increasing the cellular levels of p53. Current Biology Dispatch R625

generate the immunoglobulin and T-cell receptor reper- 13. Koonin EV, Altschul SF, Bork P: Functional motifs. Nat Genet 1996, 13:266-268. toire or that are involved in the switching of immunoglobu- 14. Callebaut, I, Mornon JP: From BRCA1 to RAP1: a widespread BRCT lin isotypes. The predisposition to malignancy, especially module closely associated with DNA repair. FEBS Lett 1997, in lymphoid cells, might be explained by the accumulation 400:25-30. 15. Nash RA, Caldecott KW, Barnes DE, Lindahl T: XRCC1 protein of unrepaired chromosomal damage, leading to the dele- interacts with one of two distinct forms of DNA ligase III. tions and translocations typical of tumorigenesis. Finally, Biochemistry 1997, 36:5207-5211. 16. Masson M, Niedergang C, Schreiber V, Muller S, Menissier-de Murcia the frequent occurrence and compromised repair of chro- J, de Murcia G: XRCC1 is specifically associated with poly[ADP- mosomal damage might make the cells of NBS patients ribose] polymerase and negatively regulates its activity following grow more slowly, or cause them to undergo apoptosis DNA damage. Mol Cell Biol 1998, 18:3563-3571. 17. Critchlow SE, Bowater RP, Jackson SP: Mammalian DNA double- more frequently, possibly producing the developmental strand break repair protein XRCC4 interacts with DNA ligase IV. defects typical of NBS — microcephaly and growth retar- Curr Biol 1997, 7:588-598. dation. Given that Mre11 is essential for mouse embryonic 18. Hofmann K, Bucher P: The FHA domain a putative nuclear signaling domain found in protein kinases and transcription stem cell viability [25], it will be interesting to know factors. Trends Biochem Sci 1995, 20:347-349. whether the NBS1–Mre11–Rad50 complex also performs 19. Stone JM, Collinge MA, Smith RD, Horn MA, Walker JC: Interaction of a protein phosphatase with an Arabidopsis serine-threonine other roles in embryonic and foetal development. receptor kinase. Science 1994, 266:793-795. 20. Ajimura M, Leem SH, Ogawa H: Identification of new genes Acknowledgements required for meiotic recombination in Saccharomyces cerevisiae. We thank John Petrini, Karl Sperling and members of the Jackson lab, espe- Genetics 1993, 133:51-66. cially Damien D'Amours, Daniel Durocher and Nick Lakin, for their helpful 21. Johzuka K, Ogawa H: Interaction of Mre11 and Rad50 – two comments during the preparation of this article. The work in our lab is sup- proteins required for DNA repair and meiosis specific double- ported by the Cancer Research Campaign. strand break formation in Saccharomyces cerevisiae. Genetics 1995, 139:1521-1532. References 22. Sharples GJ, Leach DRF: Structural and functional similarities 1. Weemaes CMR, Hustinx TWJ, Scheres J, Vanmunster PJJ, Bakkeren J, between the sbcCD proteins of Escherichia coli and the Rad50 Taalman R: A new chromosomal instability disorder the Nijmegen and Mre11 [Rad32] recombination and repair proteins of yeast. breakage syndrome. Acta Paediat Scand 1981, 70:557-564. Mol Microbiol 1995, 17:1215-1217. 2. Taalman R, Hustinx TWJ, Weemaes CMR, Seemanova E, Schmidt A, 23. Connelly JC, Leach DRF: The sbcC and sbcD genes of Escherichia Passarge E, Scheres J: Further delineation of the Nijmegen coli encode a nuclease involved in palindrome inviability and breakage syndrome. Am J Med Genet 1989, 32:425-431. genetic recombination. Genes to Cells 1996, 1:285-291. 3. Stumm M, Gatti RA, Reis A, Udar N, Chrzanowska K, Seemanova E, 24. Maser RS, Monsen KJ, Nelms BE, Petrini JHJ: hMre11 and hRad50 Sperling K, Wegner RD: The ataxia–telangiectasia variant gene 1 nuclear foci are induced during the normal cellular response to and gene 2 are distinct from the ataxia–telangiectasia gene on DNA double-strand breaks. Mol Cell Biol 1997, 17:6087-6096. chromosome 11q23.1. Am J Hum Genet 1995, 57:960-962. 25. Xiao YH, Weaver DT: Conditional gene targeted deletion by Cre 4. Jongmans W, Vuillaume M, Chrzanowska K, Smeets D, Sperling K, recombinase demonstrates the requirement for the double- Hall J: Nijmegen breakage syndrome cells fail to induce the p53- strand break repair Mre11 protein in murine embryonic stem mediated DNA damage response following exposure to ionizing cells. Nucleic Acids Res 1997, 25:2985-2991. radiation. Mol Cell Biol 1997, 17:5016-5022. 5. Matsuura K, Balmukhanov T, Tauchi H, Weemaes C, Smeets D, Chrzanowska K, Endou S, Matsuura S, Komatsu K: Radiation induction of p53 in cells from Nijmegen breakage syndrome is defective but not similar to ataxia–telangiectasia. Biochem Biophys Res Commun 1998, 242:602-607. 6. Jackson SP: Ataxia–telangiectasia at the crossroads. Curr Biol 1995, 5:1210-1212. 7. Saar K, Chrzanowska KH, Stumm M, Jung M, Nurnberg G, Wienker TF, Seemanova E, Wegner RD, Reis A, Sperling K: The gene for the ataxia–telangiectasia variant, Nijmegen breakage syndrome, maps to a 1 cM interval on chromosome 8q21. Am J Hum Genet 1997, 60:605-610. 8. Matsuura S, Weemaes C, Smeets D, Takami H, Kondo N, Sakamoto S, Yano N, Nakamura A, Tauchi H, Endo S, et al.: Genetic mapping using microcell-mediated chromosome transfer suggests a locus for Nijmegen breakage syndrome at chromosome 8q21-24. Am J Hum Genet 1997, 60:1487-1494. 9. Varon R, Vissinga C, Platzer M, Cerosaletti KM, Chrzanowska KH, Saar K, Beckmann G, Seemanova E, Cooper PR, Nowak NJ, et al.: Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell 1998, 93:467-476. 10. Matsuura S, Tauchi H, Nakamura A, Kondo N, Sakamoto S, Endo S, Smeets D, Solder B, Belohradsky BH, Der Kaloustian VM, et al.: Positional cloning of the gene for Nijmegen breakage syndrome. Nat Genet 1998, 19:179-181. 11. Stumm M, Sperling K, Wegner RD: Noncomplementation of radiation-induced chromosome aberrations in ataxia–telangiectasia/ataxia–telangiectasia-variant heterodikaryons. Am J Hum Genet 1997, 60:1246-1251. 12. Carney JP, Maser RS, Olivares H, Davis EM, LeBeau M, Yates JR, Hays L, Morgan WF, Petrini JHJ: The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double- strand break repair to the cellular DNA damage response. Cell 1998, 93:477-486.