April 2001
DNA is continually subject to the threat of damage by a widevariety of external agents as well as by spontaneous endogenousprocesses (Friedberg et al., 1995). A given agent or process can produce a complex variety of DNAlesions: for example, ionizing radiation generates free radicals thatmake single and double strand breaks, destroy deoxyribose residues andinduce numerous base alterations (Frimmer et al., 1976). DNA damage can be classified in several ways. One broad categoryconsists of base modifications, including chemical alterations,covalent joining between adjacent bases (the predominant lesion is theUV-induced pyrimidine-pyrimidine dimer (see nucleotide excision repair) and base loss. These damages to DNAtemplates can result in misincorporation during replication and canlead to heritable changes in DNA sequence. Another type of damage isintrastrand cross-linking, which can prevent DNA replication andtranscription. This review focuses on the repair of a third type ofDNA damage, DNA strand breaks, and more particularly, DNA double-strand breaks (DSBs). DSBs may occur as a result of exposure to suchexogenous factors such as radiation or chemical DNA-damaging agents.DSBs can also arise as programmed developmental modifications, such asimmunoglobulin rearrangement or in theinitiation of meiotic recombination. There is also growing evidencethat DSBs can form when DNA polymerases stall at natural replicationpause sites or when they encounter other types of DNA damage (reviewedin Rothstein et al., 2000). Since DSBs may be lethal for a cell,organisms have developed several repair pathways.
Figure 1Modes of DSB repair and some chromosomal consequences of faulty repairrepair (see also " An introduction to chromosomalaberrations).Homologous Repair(a) Precise homologous recombination with the sister chromatidrestores the original sequence.(b) Recombination between allelic sequences on homologous chromosomescan lead to Loss Of Heterozygosity if a damaged wild type sequence isrepaired using a mutant allele as a template.(c) A recombination event between two homologous sequences located innon equivalent positions on sister chromatids can give rise to anunequal exchange. In this example, one chromatid has lost one of tworepeated sequences and the other has gained a repeat.(d) When a DSB is repaired by an exchange between two homologoussequences located on different chromosomes, a reciprocal translocationcan result. Non Homologous End Joining (e) Following processing of a DSB, a rejoining event can lead to anintra-chromosomal deletion.(f) Ligation of two DNA ends located on different chromosomes resultsin a non reciprocal translocation.
An important DSB repair pathway is Homologous Recombination(HR), which is potentially a very precise mode of repair. When ahomologous sequence at the equivalent position on a sister chromatidis used as a template for repair, the original sequence of the brokenchromosome can be restored (Jablonovich et al., 1999) (Figure 1a). Recombinational repair may also utilize allelicsequences (i.e., at equivalent positions on homologous chromosomes) inthe resolution of DSBs (Figure 1b). Although recombination betweenallelic sequences is not mutagenic per se, this mode of repair can beassociated with another hazard: loss of heterozygosity (LOH).Recessive mutations may be uncovered in a heterozygote, if a damagedwild type allele is lost and a second copy of a mutant allele isconcomitantly gained (Lasko et al., 1991). Another unfavorable outcome of HR is chromosomal rearrangement,which can happen if recombination occurs between homologous sequencesat non allelic loci. This possibility arises because a large fractionof mammalian genomes consists of numerous classes of repetitive DNAsequences distributed among numerous loci. (For example, in humancells there are approximately 106 Alu elements.) If an exchange takesplace between repeats at non equivalent positions on sisterchromatids, deletions or expansions can result (Figure 1c). Exchangesbetween repeats on different chromosomes can give rise totranslocations (Figure 1d).
The principal DSB repair mechanism in higher eukaryotes is NonHomologous End Joining (NHEJ), which entails the simple ligation oftwo DNA ends with little or no homology to one another (Karran, 2000;Lewis and Resnick, 2000). Ligation can precisely rejoin two DNA ends so as introduce nochanges in sequence. However, NHEJ is most often accompanied byremodeling or processing of the ends before the rejoining event.(Figure 1e). The occurrence of several DSBs in the cell can lead tothe joining of DNA ends that are located on two different chromosomes(Figure 1f). Thus, NHEJ is relatively error-prone and is frequentlyassociated with genomic rearrangements such as deletions andtranslocations.
In this review, we focus on the pathways and enzymology of DSBmain mechanisms that cells use to repair DSBs. Many insightsconcerning DSB repair originated in studies of meiotic and mitoticrecombination in the yeast Saccharomyces cerevisiae. Therefore, webegin with a discussion of the DSB repair model as it applies tomeiotic HR as a means to introduce many of the key proteins implicatedin DSB repair in higher organisms. We then describe mitotic HR andNHEJ in yeast and mammalian cells, emphasizing both parallels anddifferences between the two systems. We also outline some of thecellular responses to DNA damage. And finally, we describe severalhuman diseases for which DSB repair defects are responsible.
Meiotic DSB repair in yeast provides a model to illustrate HR
Much of what is known about the mechanism of the repair of DSBs by HRis derived from genetic and biochemical studies of meioticrecombination in the yeast Saccharomyces cerevisiae. Yeast offers manyadvantages for the study of DSB induction and repair, including theease of synchronization of cells for simultaneous entry into themeiotic cell cycle and the relative facility of designing screens todetect mutations that affect a given point of the process. Also, DNAintermediates in the repair process can be isolated and characterized.In yeast, meiotic recombination is initiated by programmed DSBs atnumerous sites throughout the genome (Baudat and Nicolas, 1997; Gertonet al., 2000; Kleckner, 1996; Roeder, 1997; Smith and Nicolas, 1998). The repair of these breaks establishes connections (crossovers)between homologous chromosomes. Crossovers are critical to the properdisjunction of homologs from one another in the first of the twomeiotic divisions. They are also responsible for the reciprocalexchange of sequences between homologous chromosomes, therebycontributing to genetic variation. In yeast and in higher organisms,failures to create DSBs, or defects in the repair of DSBs, lead torandom disjunction of chromosomes, hence aneuploidy and gameteinviability (Bascom-Slack et al., 1997). Mammalian homologs have been identified for many of the componentsof the yeast meiotic DSB repair pathway, which underscores thelikelihood that observations concerning the mechanism of DSB-initiatedmeiotic recombination in yeast are applicable to human cells (Table1). Similarly, as exemplified by the homology of the the yeast DSBrepair protein Rad51 (and with Rad51 homologsfound in humans and other higher organisms) with the bacterial strandexchange protein RecA, the yeast meiotic DSB repair pathway representsa eukaryotic adaptation of primordial bacterial and archaebacterialrepair systems (Ogawa et al., 1993a; Ogawa et al., 1993b; Sandler etal., 1996).
Table 1: The DSB repair factors in yeast and their human homologs.
Figure 2Meiotic DSB repair in yeast. An overview of meiotic recombination between two homologouschromosomes, based on the DSB repair model, showing intermediates andthe proteins implicated in their formation by genetic and/or molecularcriteria. The points at which several proteins act is still underinvestigation, and some may be required at several steps. Proteinsthat function in both mitotic and meiotic HR are indicated in bold;all others are unique to meiosis.A DSB is introduced in a DNA duplex by the Spo11 nuclease, likelyacting in conjunction with several other proteins that are known to berequired for DSB induction.The 5' ends of the break undergo 5' to 3' resection to yield 3'-OHsingle-stranded tails.3) One of these single-stranded tails invades a homologous duplex,displacing a D-loop.4) Several steps resulting in the formation of a bimolecularintermediate with double Holliday junctions follow. At this point,correction of mismatches in heteroduplex DNA can result in geneconversion.5) Resolution of the intermediate to yield a product with a parentalconfiguration of flanking sequences (non-crossover).6) Resolution to yield a crossover product.
The mechanism of meiotic DSB repair by HR can be described by amodel that was initially proposed to account for the resolution ofbroken DNA molecules in mitotic cells (Szostak et al., 1983). Many of the molecular events predicted by this model have beendemonstrated to take place during yeast meiosis (Figure 2). Shortlyafter the synchronous entry of cells into meiosis, DSBs can bedetected by Southern analysis at numerous loci, particularly thosewhich are recombinational "hotspots" - they exhibit high frequenciesof gene conversion and crossing over (Baudat and Nicolas, 1997). At least twelve genes are required for meiotic DSB formation(reviewed in Smith and Nicolas, 1998). Although biochemical functions have not been established for most ofthem, DNA cleavage is likely mediated by the Spo11 protein in asequence-independent manner (Bergerat et al., 1997; Keeney et al.,1997). Spo11 resembles archaebacterial type II topoisomerases, which createtransient DSBs by a trans-esterification reaction that involves acovalent link between the protein and the DNA substrate. Spo11homologs are phylogenetically ubiquitous, having been found in allkingdoms examined (Celerin et al., 2000; Dernburg et al., 1998; Grelonet al., 2001; Keeney et al., 1999; McKim and HayashiHagihara, 1998;Metzler-Guillemain and de Massy, 2000; Romanienko and CameriniOtero,1999; Shannon et al., 1999; Tokuyama and Tokuyama, 2000). The meiotic failure of the mouse Spo11 mutant suggests that Spo11proteins have a similarly critical role in initiating meioticrecombination in higher eukaryotes, including in the human germline(Baudat et al., 2000; Romanienko and Camerini-Otero, 2000). The Rad50/Mre11/Xrs2 complex, which is involved in mitotic DSBrepair (see below), is also required for DSB formation (reviewed inSmith and Nicolas, 1998). This complex is also implicated in the next molecularly detectableevent of DSB repair, removal of the Spo11 protein and resection of thetwo DSB ends to varying extents by a 5' - 3' exonucleolytic activity.DSB processing results in the production of 3'-OH single-stranded DNAtails, which are highly recombinogenic and can invade a homologousduplex. Strand invasion requires the participation of several proteinsrelated to bacterial RecA, including Rad51, Rad55, Rad57, and Dmc1(see below). The Rad52 protein is also involved at this stage. Ofthese proteins, only Dmc1 is specific for meiotic recombination; theothers are also required for mitotic DSB repair. Human (and othermammalian) homologs of Rad51, Dmc1, and Rad52 have been found (seebelow).
An important intermediate predicted by the DSB repair model is ajoint molecule containing two Holliday junctions, and its existencehas been confirmed by two-dimensional electrophoresis (Schwacha andKleckner, 1994). This structure is produced by the Rad51-mediated invasion of ahomologous DNA duplex by a single-stranded DNA end, resulting in thedisplacement of a so-called "D-loop" which can anneal to the non-invading strand of the processed DSB. DNA synthesis primed by theinvading end drives further annealing of the D-loop to the initiatingduplex. Migration of Holliday junctions (branch migration) can extendthe tract of heteroduplex DNA (i.e., that which contains one singlestrand from each of the two recombining duplexes). Sequencedifferences in heteroduplex DNA created by strand invasion or bybranch migration can be corrected by the mismatch repair (MMR) system, which also functions in the repair ofmismatches that arise in mitotic cells. The principle components thatresolve meiotic mismatches are Msh2, Msh3 and Msh6 (which share homology withthe bacterial mismatch repair protein MutS), along with Mlh1 and Pms1 (homologs of bacterialMutL) (Borts et al., 2000). MutS-like proteins recognise and bind to DNA distortions caused bymismatches, and Mlh1 and Pms1 subsquently bind to the same sites butthe details of later stages of repair are unclear. MutS and MutLhomologs have been found in higher eukaryotes, including mice andhumans, and in both, derangements of MMR function have been associatedwith cancer development. MMR proteins may also influence the extensionand/or maturation of the heteroduplex tract.
Holliday junctions must be cleaved to produce recombinant molecules.Depending on the direction of cleavage, the resulting molecules willor will not have exchanged sequences flanking the region ofheteroduplex DNA (crossing over). This step could be performed eitherby a Holliday junction-specific resolvase or by a topoisomerase.Resolvase activity has been identified in yeast meiotic and inmammalian testis extracts (Constantinou et al., 2001) but the corresponding protein(s) have not yet been identified.Throughout the genome, the distribution of crossovers and resultingchiasmata (the cytologically detectable manifestation of crossovers)is non-random, but each pair of homologous chromosomes must experienceat least one crossover, which is probably sufficient to ensure propersegregation during the first meiotic division (Kaback, 1996). Moreover, crossovers are non-randomly distributed along chromosomes,such that a crossover at a given locus decreases the possibility of anearby event. Little is known about the molecular basis of thisphenomenon, termed "interference". Two other MutS homologs,Msh4 and Msh5 (which have no role inmismatch repair), Mlh1 and an exonuclease, Exo1, are required fornormal levels of crossing over. The Msh4 homolog has been cloned inmammals but its role in chiasma formation has not yet been determined.The murine Mlh1 protein is essential for both male and female meiosis;mlh1 meiotic cells arrest before the first division and are deficientin chiasmata.Since most of the proteins that participate in meiotic DSBrepair in yeast have homologs in higher eukaryotes, it is likely thatthe meiotic recombination pathway described for yeast is conservedthroughout evolution. In addition, most of these proteins also haveimportant roles during DSB repair in vegetatively growing cells,suggesting that there is much mechanistic overlap between meiotic andmitotic DSB repair.
Mitotic DSB repair by homologous recombination
In yeast, DSBs are repaired predominantly by HR. Although it waspreviously assumed that DSBs in mammalian cells were repaired mainlyby non-homologous mechanisms, there is growing evidence that HR issignificant for higher organisms as well (Jasin, 2000). In addition, recent results suggest that mutations in genes encodingproteins that function in HR can potentially confer a hypermutablephenotype conducive to tumorigenesis. There are several types ofhomologous repair: single-strand annealing, break-induced replicationand gene conversion, which differ with respect to the recombinantproducts they yield and with respect to their genetic control (forreview see (Haber, 2000). In post-replicative diploid cells, DSBs can be repaired by usingeither the sister chromatid or the homologous chromosome as atemplate, and the choice between the two is strongly regulated. Duringmeiotic DSB repair in yeast, genetical and physical studies have shownthat most repair events involve homologous chromosomes, rather thansister chromatids. In contrast, in mitosis, when the sister chromatidis available (after S-phase), cells are likely to choose it as therepair partner (Haber, 2000).
The Rad52 epistasis groupAs has been shown for meiotic recombination, HR in mitotic cells|349|requires the RAD52epistasis group of proteins,which includes Rad51, Rad55, Rad57, Rad52, Rad54 and Rad59 (Sung etal., 2000). Rad50, Mre11 and Xrs2 alsohave an important role in non-homologous recombination as well. Inyeast, the rad51, rad52 and rad54 mutations confer X-rayhypersensitivity and severe defects in meiotic and mitoticrecombination (Ivanov and Haber, 1997). In vertebrates, Rad51 is a key player in cell proliferation: rad51-/- knock out mice die early in development (Lim and Hasty, 1996) and rad51-/- cell lines cannot be established (Tsuzuki et al., 1996). The depletion of Rad51 from chicken DT40 cells leads to anaccumulation of chromosome breaks, cell cycle arrest and death (Sonodaet al., 1998). Human Rad51 can be seen as discrete foci in nuclei exposed toionizing radiation or radiomimetic chemicals but not to UVirradiation. Although the yeast RAD55 and RAD57 share similaritieswith RAD51, mutations in these two genes confer a less pronouncedphenotype. Furthermore, overexpression of RAD51can suppress defectsseen in the rad55 and rad57 null mutants (Hays et al., 1995). Mammalian cells have six Rad51 homologs- Rad51B,Rad51C, Rad51D, Xrcc2, Xrcc3, and Dmc1 (Liu et al.,1998; Pittman and Schimenti, 2000; Shu et al., 1999; Takata et al.,2000). Of these, Dmc1 is expressed only meiotically and likely has nomitotic function (Dosanjh et al., 1998; Pittman et al., 1998; Yoshidaet al., 1998). All of these Rad51-like proteins, however, have important roles inHR but, unlike Rad51, are not essential for cell viability. The humanand yeast Rad51 proteins initiate pairing between single-stranded DNAand duplex DNA and catalyse strand exchange (reviewed in Sung et al.,2000). The in vitro reaction mediated by yeast Rad51 is stimulated by yeastRad52, Rad54, Rad55, Rad57 and the single stranded binding proteinRPA.
In yeast, RAD52 is the most essential mitotic DSB repair gene, becauseit is required for all three types of homologous DSB repair describedabove (single-strand annealing, break-induced repair and geneconversion) (Ivanov and Haber, 1997). RAD59 shares some homology with RAD52 but mutant rad59 strains areless X-ray sensitive (Bai and Symington, 1996). RAD59-mediated repair events still require the RAD52 gene product.Rad52-/- knockout mice are viable and fertile. Murine mutant stemcells are not hypersensitive to radiation although they exhibit in HR,as measured by gene targeting (Rijkers et al., 1998). This absence of a strong phenotype in mammalian cells could be dueto a functional redundancy with Rad52 homologs not yet identified.Biochemical studies have shown that the yeast and human Rad52 proteinsfacilitate the annealing of complementary single-stranded DNAs andstimulate Rad51-mediated strand exchange, presumably by targetingRad51 to single-stranded DNAs. In addition, Rad52 appears to bindselectively to DNA ends (Sung et al., 2000). Curiously, in Drosophila melanogaster, Caenorhabditis elegans, andArabidopsis thaliana, the complete genome sequence for these organismsprovides no evidence of a Rad52 homolog.
The Rad54 protein is a member of the SWI2/SNF2 family of DNAdependent-ATPases which is involved in several aspects of DNAmetabolism, including transcription, nucleotide excision repair, andpost-replicative repair (Sung et al., 2000). Yeast rad54 mutants are more impaired in sister chromatidrecombination than in interhomolog gene conversion. One possibleexplanation is that the RAD54 related gene, RDH54/TID1, could bespecialised for inter-homolog recombination (Klein, 1997). Consistent with this proposal is the observation that animals thatare homologous for a knockout of the RAD54 gene are viable and fertilebut their cells are hypersensitive to ionizing radiation and DSB-inducing agents (Dronkert et al., 2000; Essers et al., 1997). The efficiency of gene targeting is reduced 5- to 10-fold in rad54cells. The Rad54 protein stimulates RAD51-mediated strand exchange(Sung et al., 2000). Based on the functions of other members of the SWI2/SNF2 family, ithas been suggested that RAD54 could also be involved in remodellingchromatin for repair and recombination.
BRCA1 and BRCA2The recent connection between HR and tumorigenesis comes fromobservations that Rad51 interacts with the tumor suppressor proteinp53 and the two breast cancer susceptibility geneproducts, BRCA1 and BRCA2(reviewed in Dasika et al., 1999).|20026| p53 plays a crucial role in linking cell cycleprogression to the presence of DNA lesions. Embryos that lack bothRad51 and p53 develop further than do embryos deficient for Rad51alone, and some double knockout animals can survive. This suggeststhat the failure to repair DSBs leads to death in a p53-dependentpathway. However, the nature of this interaction at the molecularlevel is not known.
More attention has been given to the link between Rad51 and the twoBRCA proteins. Mouse BRCA1 or BRCA2 knockouts show early embryoniclethality, similar to what is seen for RAD51 knockout mice (Hakem etal., 1998; Suzuki et al., 1997). A truncated BRCA2 gene can allow embryo survival but these animalsare prone to tumor development. Their cells are radiation sensitiveand exhibit spontaneous chromosomal aberrations. Loss of functionalBrca1 results in a slight sensitivity to radiation and DNA-damagingchemicals and a decrease in homologous gene targeting. A fraction ofRad51 colocalises with BRCA1 and BRCA2 in mitotic S-phase cells. AfterUV irradiation or exposure to DNA damaging agents, these threeproteins relocalize to potentially active repair sites. BRCA1 is aphosphorylated target of the ATM protein, which is a sensor of DNAdamaging agents (see below). BRCA1 also interacts with theRad50/Mre11/Nbs1 complex, which is involved in both HR, mentionedabove, and in NHEJ (see below). Little is known about the functions ofBRCA1 and BRCA2: although they could have a direct role in DSB repair,they are more likely involved in the regulation of DNA repairprocesses (see below).
Numerous other proteins that participate in mitotic HR have beendescribed. Among these, Rad50, Mre11 and Xrs2 (described below inconjunction with NHEJ) have multiple functions: HR,telomerelength maintenance, meiotic DSB induction, andcheckpoint regulation. HR events also involve nearly all thecomponents of the replication machinery. Mismatch repair proteinscorrect some of the mismatches created during repair and are involvedin DSB processing in the single-strand annealing pathway. Thematuration of recombination intermediates is performed by Hollidayjunction migration and resolution proteins.
Beyond its role in DSB repair, HR is relevant to other chromosomalmaintenance processes. In yeast, in the absence of telomeraseactivity, the HR machinery can restore telomere length (Le et al.,1999; Nugent et al., 1998). In addition, there is growing evidence that HR has a critical rolein the reinitiation of DNA synthesis at broken replication forks(Rothstein et al., 2000).
Non Homologous End Joining
NHEJ is a repair mechanism that is utilized in both lower andhigher eukaryotes, as evidenced by the conservation of many of theprotein components involved in the process. In mammalian cells, it isconsidered to be the major repair pathway since cells mutated in NHEJcomponents display significantly increased sensitivity to ionizingradiation. In contrast, in lower eukaryotes, such as in yeast, NHEJ isless prominent in DSB repair. Indeed, S. cerevisiae mutants withdefects in NHEJ proteins display increased sensitivity to ionizingradiation only in the absence of HR.In mammalian cells, much of our recent knowledge concerning thefunction of NHEJ components comes from V(D)J recombination studies(for review see (Fugmann et al., 2000).
V(D)J recombination is a programmed mechanism which assembles thegenes encoding immunoglobulin and T-cell receptors by fusing two ormore gene segments (termed V, D and J) in developing lymphocytes. Thelymphoid-specific recombination activating proteins Rag1and Rag2 initiate this reaction bycreating a DSB at recombination signal sequences, which are adjacentto the V,D and J segments. This cleavage creates two types ofrecombination intermediate, covalently sealed (hairpin) coding endsand blunt signal ends. Signal ends are generally joined withoutprocessing, producing precise signal joints. In contrast, when hairpincoding ends are joined, terminal nucleotides are frequently lost oradded, and imprecise coding joints of great variety are therebygenerated. Through the analysis of DNA repair mutants it has beenshown that many of the repair steps involved in the V(D)J reaction aredependent on NHEJ components.
Factors involved in NHEJ
Ku protein and DNA-dependent protein kinase (DNA-PKcs)The Ku protein was originally identified as an autoantigenrecognized by sera from autoimmune patients. It was subsequently shownto be a DNA end-binding complex composed of a 70 kDa (Ku70) and an 80kDa (Ku80) subunit (for review see Featherstone and Jackson, 1999). The corresponding genes in mammalian cells are XRCC5 (Ku80)and XRCC6 (Ku70). Mice containingdisruptions of both of these genes have been generated and theydisplay significant growth retardation as well as premature senescence(Gu et al., 1997a; Gu et al., 1997b; Nussenzweig et al., 1996; Zhu etal., 1996). Additionally, they are extremely sensitive to ionizing radiation andare immunodeficient, due to their inability to carry out V(D)Jrecombination. The S. cerevisiae Ku homolog consists of 70 and 85 kDasubunits (Hdf1 and Hdf2) that, like the mammalian heterodimer, exhibitDNA end-binding activity (Boulton and Jackson, 1996; Feldmann et al.,1996; Feldmann and Winnacker, 1993; Milne et al., 1996). Yeast Ku mutants are viable but are defective in NHEJ. However, theyexhibit sensitivity to ionizing radiation only if HR is not functional(i.e., in a rad52 background).
DNA-PKcs is a 460 kDa protein with a serine/threonine protein kinaseactivity that can be stimulated through an interaction with DNA ends(for review see Smith and Jackson, 1999). Studies in vitro have demonstrated that potential targets for thekinase activity of DNA-PKcs include p53,c-myc,the transcription factor Sp1, RNA polymerase IIand the Ku protein. The corresponding mammalian gene isXRCC7, and similar to what is observed forKu disruptions inmice, XRCC7 mutations confer severe immunodeficiency and radiationhypersensitivity (Gao et al., 1998; Jhappan et al., 1997; Taccioli etal., 1998). However, in contrast to Ku mutants, DNA-PKcs mutants are notcompletely defective in V(D)J recombination. While they are unable tocarry out coding joint formation, they are capable of forming signaljoints at levels similar to those formed in normal mice. This resultsuggests that there may be some differences between the roles of DNA-PKcs and Ku in the process of NHEJ. Interestingly, in S. cerevisiaethere are at present no known homologs of DNA-PKcs. Several proteinsthat share limited homology with DNA-PKcs (Tel1, Mec1) have beenidentified; however, cells containing mutations of these relatedproteins do not display any NHEJ defects.
In mammalian cells, the Ku heterodimer has been shown to interact withDNA-PKcs, the resulting complex being collectively referred to asDNA-PK. The Ku heterodimer is considered to be the regulatory subunitwhile the kinase is the catalytic component of this complex. Extensivebiochemical analysis of DNA-PK suggests that, following the formationof a DSB, the Ku heterodimer binds and recruits DNA-PKcs (Yaneva etal., 1997; Yoo and Dynan, 1999) (Figure 3).
Figure 3DSB repair by Non Homologous End Joining. A single Ku heterodimerbinds to each free DNA end of a DSB and recruits DNA-PKcs, resultingin the formation of the DNA-PK complex. Subsequently, Xrcc4 /Ligase IVcomplex binds to each of the DNA ends and interacts to form a tetramerthat may serve to bridge the DNA ends. Other repair proteins arelikely involved in this pathway, including the Mre11/Rad50/Nbs1 (Xrs2)complex.This enzymatically active complex subsequently promotesthe translocation of Ku away from the break and the recruitment ofadditional protein factors necessary for the completion of the endjoining process.
Figure 3DSB repair by Non Homologous End Joining. A single Ku heterodimerbinds to each free DNA end of a DSB and recruits DNA-PKcs, resultingin the formation of the DNA-PK complex. Subsequently, Xrcc4 /Ligase IVcomplex binds to each of the DNA ends and interacts to form a tetramerthat may serve to bridge the DNA ends. Other repair proteins arelikely involved in this pathway, including the Mre11/Rad50/Nbs1 (Xrs2)complex.
Xrcc4 and DNA ligase IVAnother factor required for NHEJ is encoded by the gene XRCC4. In mammalian cells, this gene was cloned bycomplementation of a rodent cell line (XR-1) mutation that confershypersensitivity to ionizing radiation, as well as defects in V(D)Jrecombination (Li et al., 1995). Interestingly, disruption of the gene in mice leads to embryoniclethality due to massive apopototic death in the nervous system (Gaoet al., 2000).Analysis of fibroblast cell lines derived from these embryosindicate that, similar to what is observed for Ku-deficient celllines, these cells display premature senescence, increased radiationsensitivity, and defects in V(D)J recombination. Similarly, in yeast,mutants of the XRCC4 homolog (LIF1), also have a phenotype thatclosely resembles that of Ku mutants: decreased levels of NHEJ as wellas radiation sensitivity in the absence of HR (Herrmann et al., 1998). These results strongly suggest that XRCC4 and Ku function in thesame DNA repair pathway.
XRCC4 is a ubiquitously expressed protein that appears tohomodimerize. Although the amino acid sequence provides no clues as toits function, the finding that XRCC4 interacts with DNA ligase IVsuggests that it may recruit or activate ligase IV to complete the endjoining reaction (Critchlow et al., 1997; Grawunder et al., 1997). Indeed, evidence of a functional interaction comes from studiesshowing that, similar to the effect of XRCC4 inactivation, disruptionof the DNA ligase IV gene in mice leads to embryonic lethality (Barneset al., 1998; Frank et al., 1998). However, disruption of ligase IV in an established mammalian cellline results in radiation sensitivity and a defect in V(D)Jrecombination (Grawunder et al., 1998). Overexpression of other DNA ligases in this cell line (DNA ligase Ior III) is unable to compensate for these defects, suggesting that DNAligase IV is the major if not only DNA ligase involved in end joining.A homolog has also been identified in S. cerevisiae (DNL4), and dnl4mutants have been shown to have significant defects in NHEJ (Schar etal., 1997; Wilson et al., 1997).
Rad50, Mre11 and Xrs2 (Nbs1)The Rad50/Mre11/Xrs2 complex was first identified in S.cerevisiae as a factor involved in DSB repair (for review see Haber,1998) It has been more explicitly demonstrated subsequently that thiscomplex is involved in both HR as well as in NHEJ. Interestingly, inS. cerevisiae, the only end joining events recovered in mre11 mutantsare those that do not exploit terminal homology, thus suggesting thatthe Rad50/Mre11/Xrs2 complex is specifically required for the use ofmicrohomology. Mammalian Mre11 and Rad50 homologs associate with theNbs1 protein, which is likely a functional homologof Xrs2 in higher eukaryotes (Carney et al., 1998). A truncated form of the NBS1 gene is responsible for the humanautosomal recessive disorder Nijmegan Breakage Syndrome (NBS) (Matsuura et al., 1998; Varon et al., 1998). Additionally, a truncating mutation of the MRE11 gene has beendemonstrated to be at the origin of an ataxia-telangiectasia (A-T, seebelow) related syndrome (Stewart et al., 1999). Both of these disorders are associated with genomic instability,cancer predisposition and immunodeficiency. In contrast, studies inmice indicate that a complete disruption of MRE11, RAD50 or NBS1results in early embryonic lethality (Luo et al., 1999; Xiao andWeaver, 1997; Zhu et al., 2001). Rad50 has been shown to be an ATP-dependent DNA binding protein(Raymond and Kleckner, 1993). Mre11 possesses several biochemical activities, the most relevantbeing a 3'-5' double-stranded DNA exonuclease activity that ispostulated to play a role in removing damaged or mismatched DNAtermini and exposing short lengths of single-stranded DNA (Paull andGellert, 1998; Trujillo et al., 1998).
The cellular response to DNA damage
The cellular response to DSBs is a complex process that involvesa network of interacting signal transduction pathways (for review seeDasika et al., 1999; Zhou and Elledge, 2000). This process is initiated by as yet unidentified proteins thatdetect or sense DNA damage and subsequently transmit a signal byactivating a cascade of phosphorylation events. This ultimatelyresults in the initiation of a number of cellular responses, whichhelp to ensure the maintenance of genomic stability, including cellcycle arrest, transcriptional activation, recruitment and activationof DNA repair proteins and, in some cases, induction of cell death byapoptosis (Figure 4). The importance of this response is evidenced bythe fact that mutations that alter any aspect of the process havesignificant effects on DSB repair. Indeed, several syndromesassociated with genomic instability and cancer predisposition, such asA-T and Nijmegan breakage syndrome (NBS), involve mutations in genesthat are involved in the cellular response to DNA damage.
Figure 4DNA damage response (after Zhou and Elledge, 2000). DNA damage is recognized by sensor proteins that then initiate anetwork of signal transduction pathways. This ultimately results inthe activation of effector proteins that execute the functions of theDNA damage response, including recruitment of DNA repair proteins,cell cycle arrest, damage induced transcription, or the induction ofapopotosis.
Ataxia telangiectasia Ataxia telangiectasia (A-T) is a diseaseclinically characterized by progressive neurodegeneration, facialtelangiectasia, immune deficiency, gonadal dysgenesis and cancerpredisposition (for review see (Rotman and Shiloh, 1999). A-T cells display increased sensitivity to killing by ionizingradiation and are defective in several DNA damage-induced checkpointcontrols. The product of the ATM gene (A-Tmutated) belongs to a family of protein kinases that are structurallyrelated to phosphatidylinositol 3 (PI-3)-kinases (Savitsky et al.,1995). However, the ability of ATM to function as a protein and not a lipidkinase suggests that it is involved in the signal transduction cascadethat is initiated in response to DNA damage. Indeed, Atm has animportant role in the response to ionizing radiation, byphosphorylating several key proteins such as p53,Mdm2, Chk1, Nbs1 and Brca1 in response to DNAdamage. These phosphorylation events are, in part, responsible for thecell cycle arrest that is necessary for DSB repair. Cells in whichATM is mutated are defective in the arrest at both the G1 and G2phases of the cell cycle. In addition, while normal cells exhibit adose dependent inhibition of DNA synthesis following exposure toionizing radiation, A-T cells display almost no alteration in theirrates of replication.
Nijmegan breakage syndromeNBS is characterized by clinical and cellular features thatresemble AT in many respects (for review see (Shiloh, 1997). In particular NBS patients exhibit chromosome instability, immunedeficiency, microencephaly and developmental delay. In addition,cells from such individuals display increased sensitivity to killingby ionizing radiation and as well as radio-resistant DNA synthesis. Aspreviously described, the Nbs protein forms a complex with Mre11 andRad50 that is required for the repair of DSBs by both NHEJ and HR(Carney et al., 1998). Functional analysis of this complex with immunoflourescently labeledantibodies indicates that following exposure to ionizing radiation,the Mre11/Rad50/Nbs1 complex localizes to sites of DNA damage, formingdiscrete nuclear foci early in the DNA damage response that remainassociated with DSBs until the repair is complete (Maser et al., 1997;Mirzoeva and Petrini, 2001). Interestingly, it has recently been shown that ATM directlyphosphorylates Nbs1 on several sites that are necessary for theformation of these foci (Gatei et al., 2000; Lim et al., 2000; Wu etal., 2000; Zhao et al., 2000). This finding provides a functional link between ATM and Nbs1 thatmay explain the similar phenotypes associated with the correspondingsyndromes.
The breast cancer susceptibility gene, BRCA1BRCA1 was initially identified as a tumor suppressor genemutated in a significant percentage of patients with familial breast cancer and/or (for review see (Scully and Livingston, 2000). The normal cellular function of this gene has subsequently beenshown to involve the maintenance of genomic stability through itsinvolvement in DSB repair (see above) and trancriptional regulation.The ability of BRCA1 to regulate several different cellular processesis thought to result from its association with a large proteincomplex, BASC, which contains proteins involved in repair, replicationand transcription (Wang et al., 2000). Following DNA damage, BRCA1 undergoes phosphorylation by both theAtm and Chk2 kinases (Cortez et al., 1999; Lee et al., 2000). This results in the subsequent dissociation of BRCA1 from itspartner CtIP, a protein of unknown function that associates with thetranscriptional repressor CtBP (Li et al., 1999). It is thought that dissociation of BRCA1 allows it to subsequentlyactivate transcription of DNA-damage-response genes such asp21 and GADD45.ConclusionsSaccharomyces cerevisiae has been an outstanding model organism forstudying the genetics and biochemistry of recombination processes. Theextensive work that has been carried out with this organism has led toa detailed mechanistic understanding of DSB repair. The relavance ofthese studies to the comprehension of DSB repair in higher eukaryotesis supported by the significant conservation of genes involved in thisprocess. However, the emergence of various tumor suppressors aspotential modulators of recombination emphasizes the additionalcomplexity of DNA repair and its consequences in mammals. Anotherrecent advance in the field has been the discovery of numerousendogenous sources of DSBs. 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Conclusions
Saccharomyces cerevisiae has been an outstanding model organism forstudying the genetics and biochemistry of recombination processes. Theextensive work that has been carried out with this organism has led toa detailed mechanistic understanding of DSB repair. The relavance ofthese studies to the comprehension of DSB repair in higher eukaryotesis supported by the significant conservation of genes involved in thisprocess. However, the emergence of various tumor suppressors aspotential modulators of recombination emphasizes the additionalcomplexity of DNA repair and its consequences in mammals. Anotherrecent advance in the field has been the discovery of numerousendogenous sources of DSBs. These lesions can be generated duringspecific programmed processes such as V(D)J recombination and meiosis,as byproducts of normal cellular metabolism - for example duringreplication or transcription - as well as by environmental factorssuch as ionizing radiation, radiomimmetic drugs or reactive oxygenspecies. Thus, DSBs are lesions with which the cell is frequentlyconfronted, and improper or inefficient repair can be extremelydetrimental at both the cellular and organismal levels. This is madeparticularly evident by the wide-ranging defects of knockout micelacking important DSB repair genes and by the severity of humandiseases (i.e. Ataxia telangiectasia, Nijmegan breakage syndrome)associated with loss of function of DSB repair genes.
Atlas of Genetics and Cytogenetics in Oncology and Haematology
Tying up Loose Ends: Generation and Repair of DNA Double-Strand Breaks
Online version: http://atlasgeneticsoncology.org/deep-insight/20008/tying-up-loose-ends-generation-and-repair-of-dna-double-strand-breaks