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GDA213.QXD 03/22/2000 02:27 Page 144 144 DNA double strand break repair in mammalian cells Peter Karran Human cells can process DNA double-strand breaks (DSBs) by either homology directed or non-homologous repair pathways. Defects in components of DSB repair pathways are associated with a predisposition to cancer. The products of the BRCA1 and BRCA2 genes, which normally confer protection against breast cancer, are involved in homology-directed DSB repair. Defects in another homology-directed pathway, singlestrand annealing, are associated with genome instability and cancer predisposition in the Nijmegen breakage syndrome and a radiation-sensitive ataxia-telangiectasia-like syndrome. Many DSB repair proteins also participate in the signaling pathways which underlie the cell’s response to DSBs. Addresses Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, UK EN6 3LD; e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:144–150 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations A-T ataxia telangiectasia ATM ataxia telangiectasia mutated ATR ataxia telangiectasis Rad3-related DNA-PK DNA-dependent protein kinase DNA-PKcs DNA-PK catalytic subunit DSB double-strand break HR homology directed recombination MMR mismatch repair NHEJ non-homologous end joining PCNA proliferating cell nuclear antigen RPA replication protein A SCID severe combined immune deficiency SSA single-strand annealing TCR transcription-coupled repair V(D)J variable division joining Xrcc4 X-ray cross-complementing 4 Introduction DNA double-strand breaks (DSBs) are a common form of DNA damage and DSB rejoining is a fundamental mechanism of genome protection. Breaks arise through direct action of ionizing radiation or some chemicals, and indirectly as a product of blocked replication forks. The ability to repair DSBs — and to ensure that repair is performed with appropriate fidelity — is a fundamental part of genome protection. The three known DSB repair pathways are outlined in Figure 1 and the participating proteins listed in Figure 2. The pathways are conserved between Saccharomyces cerevisiae and mammalian cells although the relative importance in each differs considerably. The prevailing view has been that homologous recombination is the predominant pathway of DSB repair in yeast whereas mammalian cells presented with similar substrates use non-homologous or illegitimate pathways. This still seems to be the consensus although there is mounting evidence that DSB repair via homologous recombination in mammalian cells is more significant than was previously thought [1•,2•]. In addition, there are indications that some proteins may participate in more than one of the three repair pathways. Non-homologous end rejoining Non-homologous end rejoining (NHEJ) has been considered the major pathway of DSB repair in mammalian cells (Figure 1). Repair is achieved without the need for extensive homology between the DNA ends to be joined. NHEJ processes the site-specific DSBs introduced during V(D)J (variable [division] joining) recombination and its importance is emphasised by the severe combined immune deficiency (SCID) and ionizing radiation sensitivity of mice with NHEJ defects. NHEJ involves a DNA end-binding heterodimer of the Ku70 and Ku80 proteins which activates the catalytic subunit (DNA-PKcs) of DNA-dependent protein kinase (DNA-PK) by stabilising its interaction with DNA ends. This facilitates rejoining by a DNA ligase IV/Xrcc4 (X-ray cross-complementing 4) heterodimer. (For a recent review of DNA-PK, see [3].) The presence of constitutively high steady-state levels of DNA-PK is consistent with a role as a primary DNA damage recognition factor. The family of kinases to which DNA-PKcs belongs contains the important ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia Rad3-related) signalling proteins. All three proteins are serine/threonine protein kinases which have some sequence similarity to phosphatidylinositol kinases. DNA-PK can phosphorylate p53 but the p53 response is apparently intact in DNA-PK-defective mouse cells which express wild-type p53 [4•]. This rules out DNA-PK as an essential requirement for activation of the p53 DNA damage response. Other plausible targets for DNA-PK include the singlestranded binding protein RPA (replication protein A), the DNA ligase IV cofactor Xrcc4, Ku, and DNA-PKcs itself. It is noteworthy that, although the stimulation of DNA ligase IV activity by phosphorylated and unphosphorylated forms of Xrcc4 is similar, phosphorylation of Xrcc4 may prevent its direct association with DNA [5•]. This could provide a means to regulate rejoining by modulating the interactions among Xrcc4, DNA and DNA ligase IV. Both Ku subunits and DNA-PKcs are essential for repair by NHEJ although defects in DNA-PKcs generally confer a milder phenotype than defects in Ku. DNA-PK activity is undetectable in Ku-defective cell lines, indicating that DNA binding by the Ku70:80 heterodimer is essential for its activation. The carboxy-terminal portion of Ku80 is also important for DNA-PK function and distinct regions are responsible for Ku70 interaction and DNA-PKcs activation. Deletion of the carboxy-terminal region of Chinese hamster Ku80 imparts a phenotype similar to DNA-PK deficiency — even when high levels of DNA-PKcs are GDA213.QXD 03/22/2000 02:27 Page 145 DNA double strand break repair in mammalian cells Karran 145 Figure 1 Pathways of DSB repair. The termini of a DNA DSB introduced by ionising radiation or other means are bound either by the Ku heterodimer/DNA-PKcs complex or by hRad52. In the NHEJ rejoining pathway, repair is completed by DNA ligase IV and XRCC4. DNA strand invasion of the intact sister chromatid, facilitated by hRad51, initiates repair by homologous recombination. Resection and annealing of short regions of complementary sequence initiates repair by the SSA pathway in which ligation is preceded by the trimming of noncomplementary single-stranded DNA tails. The scheme is based on [15]. present [6•]. The requirement for this carboxy-terminal region in kinase activation is consistent with the absence of an analogous Ku80 carboxy-terminal tail in S. cerevisiae which also lack a homologous DNA-PKcs protein. pass [7•]. These findings are consistent with DNA-PKcs mediating the alignment of short stretches of singlestranded DNA prior to ligation. This would be in addition to any role of DNA-PK in signalling. The molecular architecture of DNA-PKcs suggests a structural role for the protein in the rejoining process. It has a potential DNA-binding groove and an enclosed cavity with three apertures through which single-stranded DNA could Repair by NHEJ is completed by the DNA ligase IV/Xrcc4 complex. Mice with targeted inactivation of either component exhibit embryonic lethality [8,9,10•] Consistent with their joint participation in the NHEJ pathway, the Figure 2 Proteins involved in DSB repair. The circle on the left contains a list of the known participants in NHEJ. Gene products involved in DSB repair by homology directed recombination (HR) are indicated in the large circle to the right. The SSA pathway which requires some degree of homology between joined termini is depicted as a subpathway of HR. Possible overlaps with the human excision (TCR) and MMR pathways are indicated although it should be noted that there is at present no direct indication of involvement of hMSH2 or hMSH3 in the human SSA pathway. GDA213.QXD 03/22/2000 146 02:27 Page 146 Chromosomes and expression mechanisms sensitivity of DNA ligase IV or Xrcc4 knockout mouse cells to ionising radiation, and their defects in V(D)J recombination mimic those of Ku null mice. As Ku and DNA-PKcs deficient mice are viable, however, there appears to be an additional requirement for DNA ligase IV and Xrcc4 during embryonic development. Reduced NHEJ activity may confer radiosensitivity in the absence of overt defects in immune function. Radiation sensitivity and a modest defect in DSB repair, but no significant impairment in V(D)J recombination, are associated with a mutated DNA ligase IV in cells from a radiation-sensitive leukaemia patient [11•]. The absence of a detectable effect on V(D)J recombination indicates that an alternative DNA-rejoining activity might partially substitute for defective Xrcc4/DNA ligase IV in these cells. The occurrence of a DNA ligase IV defect in a leukaemia patient suggests that reduced NHEJ might be a factor in the incidence of this disease. Homologous recombination S. cerevisiae has provided a useful model for homology directed recombination (HR) processes. HR is performed by the RAD52 epistasis group of proteins which includes the products of RAD50–55, RAD57, and RAD59, MRE11 and XRS2 (for a recent review of HR, see [12]). Although mammalian cells are considered to rely less on HR, they do perform mitotic recombination and preferentially repair DSBs by HR in late S and G2 phases of the cell cycle when an undamaged sister chromatid is available. Indeed, the DSB repair defect of murine scid (severe combined immunodeficiency) cells is only apparent in G1/early S phases [13•]. The mammalian Rad51 protein is a homolog of the Escherichia coli RecA protein and is involved in both meiotic and mitotic recombination. The S. cerevisiae and human Rad51 proteins catalyse strand exchange in a reaction which is stimulated by Rad52 and RPA [14,15]. Human Rad52 has a DNA double-strand end binding activity like Ku [16•] and it probably co-operates with hRad51 in DSB repair but this is unlikely to be the only important function of Rad51. Targeted inactivation of mRad51 results in early embryonic lethality [17•] whereas Rad52–/– mice are viable and fertile [18•]. Rad52–/– murine stem cells, although deficient in recombination, are not detectably hypersensitive to the presence of DSBs. Animals which are null for a third member of the RAD52 family, mRad54, are also viable and fertile but, in this case, their cells are hypersensitive to DSB-inducing agents [19•]. Unravelling the biochemical functions of these mammalian RAD52 family members — and defining the essential functions of the Rad51 and Rad54 proteins — is clearly a high priority. At present, it is clear that they are not functionally identical to their yeast counterparts. Human Rad51 forms discrete foci in the nuclei of cells exposed to ionising radiation (but not UV) or chemicals. In rodents, DNA damage-induced mRad51 focus formation requires mRad54 [20•]. As both mRad51 and mRad54 null cells are sensitive to ionising radiation, the direct participation of mRad54 and mRad51 in DSB repair is implied. Human cells are known to express two other Rad51-related proteins — Xrcc2 and Xrcc3 — both of which interact with Rad51 and influence DSB repair by HR [21•,22•]. Confusingly, although mRad54-null and Xrcc3-deficient cells are both sensitive to ionising radiation, only the latter are cross-sensitive to UV light. An important recent development has been the realisation that the products of the human breast cancer susceptibility genes BRCA1 and BRCA2 are involved in DNA repair. In fact, the carboxy-terminal region (BRCT domain) of these proteins defines a common motif among DNA-repair proteins [23]. Loss of functional Brca1 results in sensitivity (albeit slight) to radiation and DNA-damaging chemicals [24•–26•]. A fraction of hRad51 colocalises with Brca1 and Brca2 in mitotic cells. Following DNA damage, the three proteins are relocated to structures which also contain PCNA (proliferating cell nuclear antigen) and may represent sites of active repair [27]. Both Brca2–/– and Brca1–/– mouse fibroblasts develop spontaneous chromosome aberrations consistent with the participation of these proteins in the repair of spontaneous DSBs [28•]. Brca1 is phosphorylated by the ATM protein [26•]. ATM, which is mutated in the radiationsensitive disorder ataxia-telangiectasia, is a candidate primary sensor of certain types of DNA damage, particularly DSBs induced by ionising radiation. Brca1 in therefore a target for a key protein which signals the response to ionising radiation. Brca2–/– cells are also sensitive to DNA-damaging agents, although, curiously, display a much more pronounced sensitivity to UV than to γ-radiation. In addition, the massive sensitisation to mitomycin C which accompanies loss of Xrcc2 or Xrcc3 is not seen in the Brca2–/– cells [28•]. The likely involvement of Brca1 in HR, which is suggested by its association with mRad51, is supported by the observation that there is relatively more DSB repair by non-homologous pathways in Brca1–/– cells [2•]. This is unlikely to be the full story, however, because Brca1 also displays a DNA-damage-dependent association with the hRad50/Mre11/Nbs1 complex (see below) [25•]. This important tumour suppressor therefore appears to participate in the two pathways of DSB repair. Brca1 is also implicated in the removal of oxidised DNA bases from DNA. Damage is selectively removed from the transcribed DNA strand by a process known as transcription-coupled repair (TCR). Brca1-null cells are deficient in the TCR of DNA thymine glycol produced by ionising radiation or hydrogen peroxide but perform TCR of UVinduced DNA damage normally [24•,29•]. It is unclear whether Brca1 participates directly in TCR or indirectly via a signalling role. The involvement of Brca1 suggests GDA213.QXD 03/22/2000 02:27 Page 147 DNA double strand break repair in mammalian cells Karran that the TCR of oxidised bases may represent a form of recombinational repair. Single-strand annealing The single-strand annealing (SSA) pathway of DSB rejoining (Figure 1) can be considered as a subpathway of HR. Although it does not involve formation and resolution of Holliday junctions, SSA relies on regions of homology with which to align the strands of DNA to be rejoined. The S. cerevisiae genes which define SSA belong to the Rad52 epistasis group of HR. Their human counterparts, hRad50, hMre11 and Nbs1 (also known as p95 or Nibrin), define the human SSA pathway (Figure 2). Rad50 is an ATP-dependent DNA-binding protein. Mre11 has several activities, the most important of which may be a 3′→5′ double-stranded DNA exonuclease which may remove damaged or mismatched DNA termini and expose short lengths of single-stranded DNA. This initiates a search for short regions of homology between the fragments to be joined. If the sister chromatid is available, the single-stranded tails may invade it to initiate rejoining by the HR pathway. Alternatively, sites of limited homology within the resected region may anneal to begin repair by SSA. The Nbs1 protein is functionally homologous to the S. cerevisiae Xrs2 protein. The NBS gene, which is located on chromosome 8q21, is mutated in patients with the Nijmegen breakage syndrome (NBS: for a recent review, see [30]). Cells from NBS patients exhibit many features associated with DSB-repair deficiency: sensitivity to ionising radiation, immune deficiency, chromosome instability, and a predisposition to lymphoreticular tumours. The pathway which is impaired in these cells clearly provides significant protection against the effects of unrepaired DSBs introduced either by external agents or through normal metabolic processes. Despite this, and in contrast to NHEJ-defective cells, there is no overt defect in DSB repair in NBS cells. Two families with truncating or missense mutations in hMRE11 have been reported [31•]. The similarity of their clinical symptoms to the cancer-prone ataxia telangiectasia (A-T) syndrome emphasises the importance of the SSA pathway. The properties of cells from affected individuals closely resemble those of NBS cells. As with NBS cells, there is no detectable defect in DSB rejoining in hMRE11-defective cells despite their ionising radiation hypersensitivity and increased spontaneous and radiationinduced chromosome instability. The effects of null mutations in mRad50 or mMre11 are severe. Animals bearing homozygous disruptions of the mRad50 gene exhibit early embryonic lethality [32•]. Embryonic mRad50–/– cells are extremely sensitive to ionising radiation and die during normal in vitro culture. Similarly, Mre11-null mouse cells are inviable and chicken cells in which Mre11 is conditionally disabled undergo proliferative arrest [33•]. These same cells sustain high levels 147 of chromosome aberrations and have a reduced capacity for repair by homologous recombination. The differential severity of Nbs1 and Rad50/Mre11 deficiency may reflect the overlapping involvement of the mammalian Rad50/Mre11 complex in different sub-pathways of DSB repair or in cellular signalling. The hRad50 and hMre11 proteins form an immunoprecipitable complex and colocalise in discrete nuclear foci following irradiation of cells. hRad50/hMRE11 foci are considered to be sited at DSBs. Focus formation requires that both proteins are phosphorylated [34•]. Their phosphorylation is dependent on functional Nbs1 and hMRE11 and cells defective in Nbs1 or expressing mutated hMRE11 do not form foci [31•,35•]. In addition to its targeting function, Nbs1 association with the hRad50/ hMre11 complex has a significant effect on the nuclease activity of the complex. It stimulates the endonucleolytic cleavage of hairpin loops, suggesting that there might be a greater overlap between the SSA, NHEJ and homologous recombination pathways than was previously recognised [36•]. It may be significant in this regard that translocations involving chromosomes 7 and 14 — which harbour immunoglobulin and T cell receptor genes, the substrates for NHEJ — are commonly observed in NBS, A-T, and A-T-like lymphocytes [30,31•]. Incorporation of Nbs1 into the hRad50/hMre11 complex also stimulates ATP-dependent endonucleoytic cleavage at a single-strand/duplex DNA junction [36•]. This activity may be significant because it represents a way of removing single-stranded DNA tails. The single-stranded tails created by resection and annealing of overlapping DNA ends are trimmed before ligation. This is most probably performed by the structure-specific endonuclease activity of the XPF/Ercc1 complex which is the human counterpart of the S. cerevisiae Rad1/Rad10 complex. In addition to Rad1/Rad10, clipping of the non-homologous 3′-single-stranded regions also requires the yeast Msh2 and Msh3 proteins, at least when the tails are relatively short [37,38•]. This represents a second function for Msh2 and Msh3. An Msh2/Msh3 heterodimer initiates the correction of some mismatches by binding to DNA containing short singlestranded loops (for a recent review of human mismatch repair (MMR), see [39]). This is another example of overlap between DNA repair pathways and the apparent dual roles of some repair proteins. The overlap does not appear to extend further and downstream participants in MMR, such as the Pms1 and Mlh1 proteins, are not required for SSA. Indeed, the Msh2 functions which are required for MMR are at least partially distinct and some mutated Msh2 proteins which are inactive in MMR retain full recombinational activity [40]. Human MMR proteins certainly act as editors of recombination. The extent to which they participate in the processing of DSBs by the SSA pathway is clearly an important area for further investigation. GDA213.QXD 03/22/2000 148 02:27 Page 148 Chromosomes and expression mechanisms Cellular signalling functions and DSB repair Many of the participants in DSB repair — including hMRE11, hRad50, DNA-PK, Ku and Xrcc4 — are phosphoproteins. Brca1 and Brca2 are both phosphorylated in a cell-cycle-specific fashion and the former is a target for the ATM protein kinase [26•]. Detailed description of signalling in the response to DNA damage is beyond the scope of this review but ATM and the tyrosine kinase cAbl protein appear to be central to the control of DSB repair. The c-Abl protein is itself activated by phosphorylation. This appears to be achieved by its interaction with the ATM protein [41]. Human Rad51 is phosphorylated by c-Abl, possibly via the formation of a tripartite complex with the ATM protein [42•]. ATM/c-Abl-dependent phosphorylation promotes the interaction between hRad51 and hRad52, suggesting that the HR recombination repair pathway is subject, at least in part, to fine control by these interactions. c-Abl is also implicated in activation of c-Jun amino-terminal kinase. Significantly, this activation is impaired in Nbs1 (and hMre11) as well as ATM-defective human cells. The ATM protein is implicated in the p53-related DNAdamage response. p53 plays a crucial role in determining the fate of cells in which DSBs persist. In particular, the embryonic lethality of both the Rad51 null [17•] and Brca1 null [29•] animals is attenuated in a p53–/– background and some double knockout animals survive to full term. This suggests that the massive failure of cellular proliferation, which is a feature of Rad51–/– and Brca1–/– embryos, is to some degree a direct consequence of an active p53. Put another way, cells which fail to repair DSBs by the Rad51and Brca1-dependent HR pathways die in a p53-dependent fashion. Promoting the deletion of cells which might otherwise perform mutation-prone DSB repair may be a facet of p53’s role as ‘guardian of the genome’. Conclusions and outstanding questions The above speculation notwithstanding, there is a clear need to define the details of the connection between DSBs, their repair or persistence, and cellular responses such as cell-cycle checkpoints and cell death/apoptosis. DSBs may be introduced by several different routes. These include direct introduction by agents such as ionising radiation or inhibitors of DNA topoisomerase II. DNA replication itself carries an intrinsic probability of DSB formation and human cells are estimated to sustain about ten DSBs per cell cycle [43]. This may be increased by the reduced availability of deoxynucleotide triphosphates or the presence of DNA lesions which either slow or block the replication fork. It is possible that the type and timing of DSBs influences the choice of repair pathway. There may perhaps be subpathways which involve slightly different constellations of, say, HR participants which are more likely to function on DSBs from a particular source. heterodimer, Rad50/Mre11 or members of the hRad52 pathway in which the winner determines the course of repair? Or is there some, more subtle means of signalling which is the appropriate pathway? It would make sense if repair by HR was activated in late S or G2 phases of the cell cycle when a homologous copy is present. How might this be achieved? Is this one of the functions of cell-cycle checkpoints or is it a simple consequence of proximity to replication forks of appropriate members of the hRad52 pathway? Detailed investigation of the signalling events which accompany the formation of DSBs in mammalian DNA is a clear priority. Acknowledgements I am grateful to Drs Bignami, West and Wood for their comments on the manuscript. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. • Sonoda E, Sasaki MS, Morrison C, Yamaguchi-Iwai Y, Takata M, Takeda S: Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells. Mol Cell Biol 1999, 19:5166-5169. Many DNA-damaging agents induce sister chromatid exchanges (SCEs). Using the chicken DT40 cell line with defined defects in HR or NHEJ, it is shown that Rad51 and Rad54, but not Ku70, affect the spontaneous or mitomycin C-induced SCE frequency. 2. Moynahan ME, Chiu JW, Koller BH, Jasin M: Brca1 controls • homology-directed repair. Mol Cell 1999, 4:511-518. The authors demonstrate that Brca1–/– mouse cells are selectively deficient in homologous recombination measured by targeted integration of transfected DNA as well as by DSB repair by homologous recombination. 3. 4. • Jimenez GS, Bryntesson F, Torres-Arzayus MI, Priestly A, Beeche M, Saito S, Sakaguchi K, Appella E, Jeggo PA, Taccioli GE et al.: DNAdependent protein kinase is not required for the p53-dependent response to DNA damage. Nature 1999, 400:81-83. The defective p53 response in the DNA-PK deficient SCGR11 cell line — which was considered to reflect the interdependence of p53 and DNA-PK — is here shown to be a consequence of a p53 mutation. In cells expressing a wild-type p53, p53 activation is demonstrated in the absence of a functional DNA-PK. This is in agreement with several other lines of evidence which indicate that p53-related checkpoint functions are activated independently of DNA-PK. 5. • Modesti M, Hesse JE, Gellert M: DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV. EMBO J 1999, 18:2008-2018. XRCC4 is shown to exhibit cooperative binding to DNA. This stimulates the adenylation of DNA ligase IV. The phosphorylated form of XRCC4 is shown to lack DNA-binding activity. 6. • Singleton BK, Torres-Arzayus MI, Rottinghaus ST, Taccioli GE, Jeggo PA: The C terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit. Mol Cell Biol 1999, 19:3267-3277. Analysis of mutations in the hamster Ku80 identifies a carboxy-terminal 178 amino acid region as being essential for stimulation of DNA-PK. This region is distinct from the one which interacts with Ku70. 7. • Leuther KK, Hammarstan O, Kornberg RD, Chu G: Structure of DNA dependent protein kinase: implications for its regulation by DNA. EMBO J 1999, 18:1114-1123. Analysis of DNA-PKcs crystallised on a lipid layer reveals an intriguing threedimensional structure. On the basis of its affinities for different DNA structures, a model for how DNA-PK might operate in DSB rejoining is proposed. 8. A related issue concerns the selection of repair pathway. Is there simply a direct competition between the Ku Smith GSM, Jackson SP: The DNA-dependent protein kinase. Genes Dev 1999, 13:916-934. Frank KM, Sekiguchi JM, Seidl KJ, Swat W, Rathburn GA, Cheng HL, Davidson L, Kangaloo L, Alt FW: Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 1998, 396:173-177. GDA213.QXD 03/22/2000 02:27 Page 149 DNA double strand break repair in mammalian cells Karran 9. Barnes DE, Stamp G, Rosewell I, Denzel A, Lindahl T: Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr Biol 1998, 8:1395-1398. 10. Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ, Sekiguchi JM, • Rathburn GA, Swat W, Wang J et al.: A critical role for DNA endjoining proteins in both lymphomagenesis and neurogenesis. Cell 1998, 95:891-902. References [6•,7•,8] together implicate mouse DNA ligase IV and XRCC4 in an essential developmental function. Animals defective for either gene exhibit embryonic lethality with a characteristic impairment of central nervous system development as well as the anticipated defect in V(D)J recombination. The inviability of these mice contrasts with the viability of Ku or DNA-PKcs mutant mice which strongly suggests an additional role for DNA ligase IV/XRCC4. 11. Riballo E, Crichlow SE, Teo S-H, Doherty AJ, Priestly A, Broughton B, • Kysela B, Beamish H, Plowman N, Arlett CF et al.: Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient. Curr Biol 1999, 9:699-702. A human cell line from a radiation-sensitive leukaemia patient is shown to have a mutated DNA ligase IV with a severely limited ability to undergo adenylation. Yeast cells expressing the mutated ligase are extremely sensitive to ionizing radiation and deficient in NHEJ in vitro. 12. Baumann P, West SC: Role of the human RAD51 protein in homologous recombination and double-stranded break repair. Trends Biochem Sci 1998, 23:247-251. 13. Lee SE, Mitchell RA, Cheng A, Hendrickson EA: Evidence for DNA • PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle. Mol Cell Biol 1997, 17:1425-1433. DNA-PK activity is here shown to be maximal in the late G1/early S and G2 phases of the cell cycle. The radiation-sensitivity and DSB rejoining defect of mouse scid cells is apparent only in the G1/early S phase. The absence of DNA-PK in the G2 phase does not affect radiation sensitivity or rejoining. 14. New JH, Sugiyama T, Zaitseva E, Kowalczylkowski SC: Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature 1998, 391:407-409. 15. Benson FE, Baumann P, West SC: Synergistic actions of Rad51 and Rad52 in recombination and DNA repair. Nature 1998, 391:401-404. 16. Van Dyck E, Stasiak AZ, Stasiak A, West SC: Binding of double • strand breaks in DNA by human Rad52 protein. Nature 1999, 398:728-731. Human Rad52 protein is shown to be an alternative DNA end binding activity to Ku. By analogy to Ku/DNA-PK in NHEJ, it is proposed that end binding by hRad52 initiates the HR repair pathways. 149 chromosome stability and protect against DNA cross-links and other damages. Mol Cell 1998, 1:783-793. The cloning of these two important RAD51 family members by complementing the extreme mitomycin C sensitivity of deficient hamster cells is described. The interaction between XRCC3/mRad51 is directly demonstrated. 22. Pierce AJ, Johnson RD, Thompson LH, Jasin M: XRCC3 promotes • homology-directed repair of DNA damage in mammalian cells. Genes Dev 1999, 13:2633-2638. The authors provide direct evidence for the participation of XRCC3 in homologous recombinational repair of DSBs. 23. Callebaut I, Mornon J-P: From BRCA1 to RAP1: a widespread BRCT module closely associated with DNA repair. FEBS Lett 1997, 400:25-30. 24. Gowen LC, Avrutskaya AA, Latour AM, Koller BH, Leadon SA: BRCA1 • required for transcription-coupled repair of oxidative DNA damage. Science 1998, 281:1009-1012. Brca1–/– mouse ES cells shown to be more sensitive to H2O2 than –/+heterozygous or +/+wild-type cells and to be deficient in the selective removal of oxidised thymine bases (thymine glycol) from the transcribed DNA strand. 25. Zhong Q, Chen C-F, Li S, Chen Y, Wang C-C, Xiao J, Chen P-L, • Sharp ZD, Lee W-H: Association of BRCA1 with the hRad50hMre11-p95 complex and the DNA damage response. Science 1999, 285:747-750. Human Rad50 is shown to complex with Nibrin and hMre11. hRad50 focus formation is reduced in a human breast carcinoma cell line with homozygous mutation in Brca1. Focus formation is restored by a transfected wild-type Brca1. Correction of the (slight) MMS sensitivity is also achieved by expressing a wild-type Brca1. 26. Cortez D, Wang Y, Qin J, Elledge SJ: Requirement of ATM • dependent phosphorylation of Brca-1 in the DNA damage response to double-strand breaks. Science 1999, 286:1162-1166. The authors provide a first indication that Brca1 might act downstream in the signalling pathway initiated by ATM. The phosphorylation of Brca1 which normally follows ionising radiation is not seen in A-T cells. Biochemical and mutational analyses confirm that Brca1 is a substrate for phosphorylation by ATM. There is some indication that this is related to the slight ionising radiation sensitivity of Brca1-defective cells. The γ-ray sensitivity of Brca1–/– cells is, however, much less pronounced than A-T cells, indicating that Brca1 is likely to be only one of the significant targets of ATM. 27. Chen J, Silver DP, Walpita D, Cantor SB, Gazdar AF, Tomlinson G, Couch FJ, Weber BL, Ashley T, Livingston DM, Scully R: Stable interaction between the products of the BRCA1 and BRCA2 tumour suppressor genes in mitotic and meiotic cells. Mol Cell 1998, 2:317-328. 17. • 28. Patel KJ, Yu VPCC, Lee H, Corcoran A, Thistlethwaite FC, Evans MJ, • Colledge WH, Friedman LS, Ponder BAJ, Venkitaraman AR: Involvement of Brca2 in DNA repair. Mol Cell 1998, 1:347-357. The authors report hypersensitivity to various DNA-damaging agents and increased rates of spontaneous chromosome aberrations in mBrca2–/– embryonic fibroblasts. Interestingly, this DNA-repair defect is associated with apparently normal cell cycle checkpoint controls. 18. Rijkers T, van den Ouweland J, Morolli B, Rolink AG, Baarends WM, • van Sloun PP, Lohman PH, Pastink A: Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation. Mol Cell Biol 1998, 18:6423-6429. mRad52–/– embryonic stem cells appear to be insensitive to DSB-inducing agents. The same cells exhibit reduced homologous recombination but the mice are viable, fertile and immunocompetent. This is in striking contrast to the properties of mRad51–/– cells and mice. 29. Cressman VL, Backlund DC, Avrutskaya AV, Leadon SA, Godfrey V, • Koller BH: Growth retardation, DNA repair defects, and lack of spermatogenesis in BRCA1-deficient mice. Mol Cell Biol 1999, 19:7061-7075. An investigation of the relationship between mBrca1 and p53. The embryonic lethality of the mBrca1 defect is partially alleviated by a p53 defect. Parallel observations are reported in cultured fibroblasts in which the poor proliferation of mBrca1–/– cells is reversed by selection for an abrogated p53 response. This is another example of p53 controlling the survival of cells with unresolved DSBs. Lim DS, Hasty P: A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol Cell Biol 1996, 16:7133-7143. A report of embryonic lethality of mRad51–/– mice and radiation sensitivity in embryonic cells. This also appears to be the first indication that the cytotoxic effects of impaired DSB rejoining may be mediated by p53. 19. 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As with A-T and NBS cells, no overt defect in DSB rejoining is observed. hMRE11/Rad50/NBS focus formation is absent in hMRE11 mutant cells which are also defective in JNK activation. The p53 response to ionising radiation serves to distinguish hMRE11 mutant cells from A-T cells. Because of the similarity of clinical phenotypes, the authors suggest that up to 6% of diagnosed A-T patients may actually harbour hMRE11 mutations. GDA213.QXD 03/22/2000 150 02:27 Page 150 Chromosomes and expression mechanisms 32. Luo G, Yao MS, Bender CF, Mills M, Bladl AR, Bradley A, Petrini JHJ: • Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionising radiation. Proc Natl Acad Sci USA 1999, 96:7376-7381. Inactivation of mRad50 is lethal in both early embryos and embryonic stem cells. Irradiation of blastocyst explants indicates a substantial sensitivity to ionising radiation in the mRad50–/– cells. 33. 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Analysis of the biochemical properties of a purified recombinant human Rad50/Mre11/Nbs complex indicates that Nbs confers several additional functions on the hRad50/Mre11 heterodimer. The authors also suggest that the tripartite complex may serve a DNA-damage recognition function which may control how DSBs are processed. This decision would be determined partly by the ability of the complex to sense differences in the type of DNA ends present. 37. Paques F, Haber JE: Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae. Mol Cell Biol 1997, 17:6765-6771. 38. Sugawara N, Paques F, Colaiacovo M, Haber JE: Role of • Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination. Proc Natl Acad Sci USA 1997, 94:9214-9219. Evidence is presented that the MSH2 and MSH3 mismatch repair proteins may participate in the removal of single-stranded DNA tails during DSB repair by SSA. 39. 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