p53: TRAFFIC COP AT THE CROSSROADS OF DNA REPAIR AND ...

REVIEWS

p53: TRAFFIC COP AT THE CROSSROADS OF DNA REPAIR AND RECOMBINATION Sagar Sengupta*‡ and Curtis C. Harris* Abstract | p53 mutants that lack DNA-binding activities, and therefore, transcriptional activities, are among the most common mutations in human cancer. Recently, a new role for p53 has come to light, as the tumour suppressor also functions in DNA repair and recombination. In cooperation with its function in transcription, the transcription-independent roles of p53 contribute to the control and efficiency of DNA repair and recombination.

MISMATCH REPAIR

(MMR). A DNA-repair process that removes mispaired nucleotides and insertion or deletion loops. NUCLEAR MATRIX

A network of nuclear proteins that provides a structural framework for organizing chromatin. NUCLEOTIDE-EXCISION REPAIR

A DNA-repair process in which a small region of the DNA strand that surrounds the UV-induced DNA damage is recognized, removed and replaced.

*Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Building 37, Room 3068, Bethesda, Maryland, 20892-4255, USA. ‡ National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India. Correspondence to C.H. e-mail: [email protected] doi:10.1038/nrm1546

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The tumour suppressor p53 functions primarily as a transcription factor and can mediate its different downstream functions by activating or repressing a large number of target genes1–4. p53 is one of the most commonly mutated genes in human cancer, which leads to the generation of a mutant protein with an altered amino-acid sequence, usually in the DNA-binding domain5,6. Cellular proteins, such as mouse double minute-2 (MDM2), and viral proteins, such as human papilloma virus-16 E6 (HPV-16 E6), can degrade wild-type p53 and limit its activity. Loss of function of wild-type p53 also occurs by the overexpression of mutant p53 due to its dominant-negative effect7. In addition to its role in transcriptional regulation, transcriptionally independent functions of wild-type p53 have a less well-known role in mediating at least some of its downstream effects, including apoptosis, DNA repair and DNA recombination. Apart from its role as a sequence-specific transcription factor, whereby p53 binds to highly conserved p53binding sequences that are generally present in the promoters of its target genes2, p53 also binds ‘nonsequence specifically’ to various DNA structures8. Analysis using various DNA-binding assays revealed that the affinity of p53 for mismatched and bulged DNA is equal to, or even greater than, that of the human MISMATCH REPAIR (MMR) complex, MSH2– MSH6, under the same binding conditions9. Perhaps more pertinently, within the in vivo context, p53 can

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bind to the NUCLEAR MATRIX. This binding affinity increases following genotoxic stress10 and is the ratelimiting factor in the repair of higher-order DNA structures11. In eukaryotes, the five main DNA-repair processes are NUCLEOTIDE-EXCISION REPAIR (NER), BASE-EXCISION REPAIR (BER), MMR, NON-HOMOLOGOUS END-JOINING (NHEJ) and 12–17 HOMOLOGOUS RECOMBINATION (HR) . Although only the transactivation-independent function of p53 is involved in HR regulation, the tumour suppressor modulates almost all other DNA-repair processes by both transactivation-dependent and -independent pathways (FIG. 1). Therefore, p53 might function as the ‘molecular node’2 that lies at the intersection of upstream signalling cascades and downstream DNArepair and -recombination pathways. In this review, we will detail the transactivation-independent roles of p53, discuss the integration of its transactivationdependent and -independent functions in DNA repair and recombination, and try to understand how the abrogation of these functions of p53 can lead to cancer in humans (summarized in TABLE 1). Nucleotide-excision repair and p53

The most versatile form of DNA repair, NER, operates on damaged bases and disrupted base pairings that are caused by ultraviolet light (UV) or oxidative damage, which leads to changes in the structure of the DNA duplex. There are two NER pathways, which have partly

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REVIEWS distinct substrate specificities. Whereas global genomic repair (GGR) scans the entire genome, transcriptioncoupled repair (TCR) recognizes lesions that are associated with transcription12,13. NER recognizes a broad range of different DNA lesions, including UV-induced

Ionizing radiation

UV, replication errors, HU

Changes in chromatin structure ATR MRN complex BRCA1 P

P

RAD9

RAD9 RPA ATRIP ATR RAD17

ATM P H2AX

P P

Lesion, adduct

ATM

CHK2

P

RAD17 ATRIP

ATM

53BP1

P

MDC1

P CHK1

P P

BLM P P

SMC1 P

P P

p53

H2AX

P

P P

53BP1

P P

NER

NER

BER Transactivationdependent processes

Transactivationindependent processes

MMR

?

BER

NHEJ MMR

HR Damage recognition Apoptosis

Cell-cycle-checkpoint arrest Apoptosis

Senescence

DNA damage

Figure 1 | p53 functions as a ‘molecular node’ in the DNA-damage response. The DNAdamage signal from DNA double-strand breaks (DSBs; left-hand pathway) or replication stress (right-hand pathway) is primarily recognized by ATM (ataxia telangiectasia mutated) or the ATR–ATRIP (ataxia-telangiectasia-and-RAD3-related–ATR-interacting-protein) complex, respectively. Changes in chromatin structure can lead to the autophosphorylation-mediated activation of ATM, which is subsequently recruited to the DSB. The site of damage contains the MRN complex (which comprises RAD50, MRE11 (meiotic-recombination-11) and NBS1 (Nijmegen breakage syndrome-1)) and BRCA1 (breast-cancer-susceptibility protein-1). H2AX (histone-2A family, member X), 53BP1 (p53-binding protein-1), MDC1 (mediator of DNA-damage checkpoint protein-1) and SMC1 (structural maintenance of chromosomes-1) are phosphorylated by ATM, and are the key proteins that are involved in transducing a DSB-induced signal. The ATR–ATRIP complex is recruited to the replication forks by the single-stranded (ss)DNA–RPA (replication protein A) complex. The RAD9–RAD1–HUS1 complex (RAD9 in the figure) and its loading factor RAD17 facilitate recognition of the stalled fork. BLM (Bloom syndrome protein) and H2AX are phosphorylated in an ATR/CHK1-dependent manner in response to replication stress. CHK1 and CHK2, the respective targets of ATR and ATM, also phosphorylate the transducer proteins. The transducer proteins transmit the signal to effector proteins such as p53, which is phosphorylated at different, yet specific, residues by ATM, ATR, CHK1 and CHK2. p53 is involved in cell-cycle arrest, DNA repair, apoptosis or senescence. p53 has roles that do not involve its transactivation functions during DNA repair — nucleotide-excision repair (NER), base-excision repair (BER), mismatch repair (MMR), non-homologous end-joining (NHEJ) — and homologous recombination (HR). The repair processes (NER, BER and MMR) are also partially influenced by the transactivation function of p53. The transactivation-independent role of p53 during apoptosis has been recently described. The continuum between the transactivation-independent and dependent functions might be dependent on the level of DNA damage and cell type. HU, hydroxyurea; UV, ultraviolet light.

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CYCLOBUTANE PYRIMIDINE DIMERS (CPDs) and pyrimidine (6-4)pyrimidone photoproducts ((6-4) PHOTOPRODUCTS). During UV damage, expression of the UV-damage DNA-binding protein (UV-DDB) — which is composed of two subunits, p127DDB1 and p48DDB2 — is induced. It has been postulated that p48DDB2 is required to activate the latent binding activity in p127DDB1, but once p127DDB1 acquires its activity, p48DDB2 is dispensable17. Xeroderma pigmentosum complementation group C (XPC)–RAD23B is a GGR-specific complex that is involved in identifying disrupted base pairings. Binding to (6-4) photoproducts by XPC–RAD23B is direct. However, binding to CPDs by XPC–RAD23B might require the prior binding of the UV-DDB complex. XPC is not required during TCR; however, during this process, the stalled RNA polymerase II must be displaced and the base damage must be specifically recognized by at least two TCR-specific factors, Cockayne syndrome group B (CSB) and CSA. The subsequent stages in GGR and TCR might be identical. Lesion recognition is followed by the binding of several other proteins: XPA and replication protein A (RPA; both of which enhance base recognition), and the transcription factor (TF)IIH subcomplex of RNA polymerase II (which unwinds DNA by the XPB and XPD helicases in the vicinity of DNA damage). The two NERspecific endonucleases, XPG and ERCC1/XPF, bind and cleave the DNA 3′ and 5′ to the site of the damage. Repair synthesis and subsequent replication (which is mediated by DNA polymerases α or ε, proliferating cell nuclear antigen (PCNA), RPA and replication factor C (RFC)) is followed by re-ligation. Patients with defective NER suffer from xeroderma pigmentosum (XP), a rare autosomal-recessive disorder that is characterized by sun sensitivity and premature malignant skin carcinomas and neoplasms. XP is genetically heterogeneous: there are eight complementation groups designated XP-A to XP-G and XP-variant (XP-V). Whereas XP-A to XP-G correspond to genetic alterations in one of seven genes that are involved in NER, XP-V is caused by defects in the post-replication-repair machinery, but NER is not impaired. Therefore, XP-V patients have normal excision repair, but show defective DNA replication after UV irradiation. This is because the gene that encodes XP-V, DNA polymerase ε, allows DNA synthesis across UV-induced TT-dimers in an error-free manner under normal conditions. Genes for XP groups A, B, D, F and G are required for both TCR and GGR, whereas genes for groups C and E are required for GGR, but not TCR17. So, does p53 affect NER in vivo? The loss of p53 in human cells results in the reduced repair of UV-induced DNA damage18–20. Intact cells that are heterozygous for mutant p53 have normal repair of (6-4) photoproducts, but decreased initial rates of CPD removal, compared with normal cells19. Therefore, it is possible that p53 affects NER in vivo by affecting the function (or functions) of the proteins that are involved in this process. For example, p53 regulates the transcription of p48DDB2 and XPC 21,22. In turn, p48DDB2 directly regulates p53 protein

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Table 1 | The effect of p53 on DNA-repair and -recombination pathways Pathway

Type of damage

Damaging agents/cause

Nucleotideexcision repair (NER)

CPD; UV; cisplatin; (6-4) photoproducts 4-nitroquinoline oxide; and other oxidative damages

Dependence on p53transactivation function

Dependence on p53transactivationindependent function

Effect of p53 on processspecific protein(s) functions

Effect of process-specific protein on p53 functions

Yes

Yes

Yes

Yes

Base-excision Single-base DNA repair (BER) damage (shortpatch BER); single-strand break (long-patch BER)

Oxidizing, methylating, alkylating, hydroxylating agents; ionizing radiation; spontaneous depurinations (shortpatch BER); X-rays (long-patch BER)

Controversial

Yes

Yes

Yes

Mismatch repair (MMR)

Mispaired nucleotides; insertion/deletion loops

Slippage of polymerase during replication of repetitive sequences or recombination; mutations in MMR genes

Yes

Yes

Yes

Yes

Nonhomologous end-joining (NHEJ)

Double-strand break

Ionizing radiation; chemical agents such as neocarzinostatin

ND

Yes

Yes

Controversial

Homologous Double-strand recombination break (HR)

Ionizing radiation; chemical agents such as neocarzinostatin

No

Yes

Yes

Yes

CPD, cyclobutane pyrimidine dimers; ND, not determined; UV, ultraviolet light.

BASE-EXCISION REPAIR

The main DNA-repair pathway that is responsible for the repair of apurinic and apyrimidinic (AP) sites in DNA. BER is catalyzed in four consecutive steps by a DNA glycosylase, which removes the damaged base; an AP endonuclease (APE), which processes the abasic site; a DNA polymerase, which inserts the new nucleotide(s); and DNA ligase, which rejoins the DNA strand. NON-HOMOLOGOUS ENDJOINING

The main DNA-repair pathway that is used throughout the cell cycle to repair chromosomal DSBs in somatic cells. NHEJ is error-prone because it leads to the joining of the breaks without a template. HOMOLOGOUS RECOMBINATION

A DNA-recombination pathway, which includes the repair of DSBs, and which uses a homologous double-strandedDNA molecule as a template for the repair of the broken DNA. CYCLOBUTANE PYRIMIDINE DIMER

(CPD). The main UV-induced lesion that functions as a structural block for transcription and replication.

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levels after UV irradiation, which implies that there are mutual regulatory interactions between these two proteins23. The recruitment of both XPC and TFIIH to the sites of CPDs and (6-4) photoproducts is facilitated by wild-type p53, but not the mutant protein24. p53inducible p48DDB2 is the key downstream factor that is responsible for the transport of XPC to the sites of DNA damage in irradiated cells25,26. This probably explains why p48DDB2 and XPC, but not p53, colocalize to the sites of UV-induced DNA damage27 (FIG. 2A). Apart from the above-mentioned transactivationdependent functions, can p53 affect NER in a transactivation-independent manner? Purified p53 does not seem to stimulate NER in a reconstituted in vitro NER assay28. However, we and others have shown that p53 can affect NER by binding to the TFIIH helicase subunits, XPB and XPD, thereby modulating their helicase activity20,29. Subsequently, it was observed that XPB and XPD helicases are also components of the p53-mediated apoptotic pathway29,30 (FIG. 2Ba). Mechanistically, mutant p53 is a less efficient inhibitor of both XPBand XPD-mediated helicase and transcription activities. So, overall it does seem that p53 affects NER in a transactivation-independent manner. As a further indication of its transactivation-independent role, p53 might function as a CHROMATINACCESSIBILITY FACTOR in NER. In response to localized subnuclear UV irradiation, after detection and initial recognition of the transcription-associated lesion (by TCR), p53-dependent chromatin relaxation occurs, which subsequently extends over the entire genome31. It is postulated that global chromatin relaxation, in turn, leads to lesion detection over the entire genome by the GGR system. Interestingly, the UV-induced chromatin


relaxation is achieved by the p53-mediated acetylation of histone H3. As p53 is not known to possess intrinsic histone acetyltransferase (HAT) activity, it recruits p300 (and probably other HATs) to NER sites to carry out the acetylation functions (FIG. 2Bb). Whereas the role of p53 in GGR is well accepted32, p53 might also facilitate TCR20,33,34, as human cells with compromised p53 function have defective TCR20,33. However, the role of p53 in TCR is controversial, as it was reported that mammalian cells that are homozygous for p53 mutations are deficient in GGR, but have normal TCR35–37. This apparent discrepancy between different studies might be due to the use of different UV sources for irradiation. It has been shown that although the loss of functional p53 significantly reduces the efficiency of TCR due to exposure to polychromatic UVB (290–324 nm), p53 is essential for GGR but dispensable for TCR when cells are exposed to ‘germicidal’, monochromatic UVC (254 nm), which is virtually absent from terrestrial sunlight38. However, the mechanistic basis for the role of p53 in UV-dependent regulation of CPD removal remains unknown. Another component of the TFIIH complex that is necessary only for TCR, CSB, interacts with p53 in vitro and in vivo 20,39. It has been proposed that the activated p53 sequesters and inactivates CSB protein, thereby stalling transcription complexes, locally blocking chromatin condensation and causing metaphase-specific fragility of human genes39. Base-excision repair and p53

BER protects mammalian cells from damage that is inflicted by cellular metabolism (including damage that results from methylating and oxidizing agents) and by spontaneous depurinations. A large number of


REVIEWS A Coactivators

Coactivators

p53

p53

p48DDB2

p53RE

XPC

p53RE p48DDB2

XPC

p48DDB2

XPC

p48DDB2

XPC

CPD

Ba TFIIH XPB

Modulates activities of XPB and XPD (in vitro)

XPD p53

b

Modulates NER

TFIIH XPB

UV

XPD Lesion

Condensed chromatin

p48DDB2

CSB

p48DDB2

Transcription-associated lesion detection XPC

p53

p300 Acetylated H3

p300 p53

p300 XPC

p53

p300 XPC

p53

XPC

Global chromatin relaxation and subsequent detection on relaxed chromatin

Figure 2 | Role of p53 in nucleotide-excision repair. A | An example of the transactivationdependent function of p53. In the presence of specific co-activators, p53 functions as a transcription factor by binding to p53-response elements (p53RE) to upregulate the expression of the xeroderma pigmentosum complementation group C (XPC) and p48DDB2 genes during nucleotide-excision repair (NER). XPC and p48DDB2 recognize and bind specific lesions on the DNA. Whereas p48DDB2 is one of the components of the ultraviolet light (UV)-induced, UV-damage DNAbinding protein (UV-DDB), XPC–RAD23B is a global genomic repair (GGR)-specific complex that is involved in identifying disrupted base pairing. UV-induced cyclobutane pyrimidine dimers (CPDs) are an example of the type of damage to which p48DDB2 transports XPC. However, physical binding between XPC and p48DDB2 is yet to be reported. B | Examples of the transactivation-independent functions of p53. p53 can modulate NER by two independent, yet possibly interlinked, mechanisms. a | XPB and XPD helicases are subunits of the transcription factor (TF)IIH and can unwind ~30 base pairs of DNA near the damaged site. In vitro, p53 binds to XPB and XPD, modulates their enzymatic functions, and thereby probably affects NER. b | p53 can also function as a chromatin-accessibility factor in vivo. During transcription-coupled repair (TCR), the UV-damage-induced DNA lesion is recognized by TCR-specific factors, such as CSB (a subunit of TFIIH), along with p48DDB2 (a subunit of UV-DDB). After the initial recognition of the transcription-associated lesion, p53-dependent chromatin relaxation occurs and extends over the entire genome, which then allows lesion detection by the NER system. During global chromatin relaxation, p53 recruits the histone acetylase p300 to NER sites to acetylate histone H3, thereby relaxing the chromatin and enhancing the detection of the lesion in the entire genome. Therefore, the presence of wild-type p53 helps in the optimal utilization of NER.


glycosylases with narrow, yet partially overlapping, substrate specificity, endonucleases and DNA polymerases are involved in this repair process. BER is mediated by at least two pathways: a ‘short-patch-repair’ pathway, which involves the repair of a single nucleotide, and a ‘long-patch-repair’ pathway, which involves repairing 2–15 nucleotides12,14. A recent study with apurinic or apyrimidinic (AP) endonuclease-1 (APE1/REF1) indicates that the transactivation function of p53 might be affected by changes in BER enzymes. APE1/REF1 associates with p53, potentiates DNA binding by p53 and enhances the transactivation, growth arrest and apoptotic functions of p53 in vivo. The downregulation of APE1/REF1 causes a reduction in the ability of p53 to transactivate its cognate promoters40. So does p53, in turn, affect BER? The first hint of an in vivo role for p53 in BER was observed using extracts from cells that expressed wild-type p53 and that had an augmented BER response41. The onset of p53 facilitation of BER in cells is determined by the level of DNA damage. Whereas low doses of γ-irradiation or the platinum compound cisplatin (which causes the formation of intrastrand crosslinks between adjacent guanines) result in an immediate enhancement of BER activity, high doses of the same DNA-damaging agents instead lead to a reduction in BER and the induction of p53-dependent apoptosis42. BER activity is associated with two distinct phases of cell-cycle progression in unstressed cells: the G0–G1 and G2–M phases. Exposure to γ-irradiation enhanced the G0–G1 BER-associated activity and attenuated the G2–M BER-associated activity. More pertinently, the alteration in BER activity after γ-irradiation is regulated by wild-type p53, but not its tumour-derived mutant forms43. Recently, Rotter and co-workers have also shown that the effect of p53 on 3-methyladenine (3-MeAde) DNA glycosylase, the first enzyme that was identified in the short-patch-repair BER pathway, is dependent on the type of stress. Wild-type p53 downregulates the transcription and activity of 3-MeAde DNA glycosylase after exposure to nitric oxide (NO), thereby preventing the creation of a mutator phenotype 44 (FIG. 3a) . By contrast, γ-irradiation elevates p53dependent 3-MeAde-DNA-glycosylase activity 44. The presence of wild-type p53 enhanced the removal of oxidized bases, such as 8-oxoG, during exposure to reactive oxygen species45 (FIG. 3b). So, is p53 absolutely required for BER or does it only have a stimulatory role in the process? A recent report indicated that BER intermediates can induce p53-independent cytotoxic and genotoxic responses46, which implies that the tumour suppressor might not have an enzymatic role in the process. However, BER is stimulated by recombinant wild-type p53 in an in vitro reconstitution system. The p53-dependent stimulation of BER is correlated with its ability to interact directly with APE1/REF1, DNA polymerase β and 8-oxoguanine glycosylase (OGG1)45,47. OGG1 has glycosylase activity that removes bases in DNA such as 8-oxoG and generates an AP site. The AP site is recognized by the


REVIEWS a DNA glycosylase

NO-induced DNA damage

p53

Mutator phenotype

DNA repair by BER

b p53

Direct interaction, enhanced activity of APE1/REF1

Interaction with DNA polymerase β, enhanced stability of polymerase

Interaction with OGG1, enhanced removal of 8-oxoG from DNA

Interaction with SeMet via APE1/REF1

Increase of 3-MeAde DNA glycosylase and OGG1 activities during γ-irradiation and ROS

BER

Figure 3 | Role of p53 in base-excision repair. a | An example of the transcription-dependent effects of p53. Exposure to nitric oxide (NO) induces p53 to cause transcriptional repression of the 3-methyladenine (3-MeAde) DNA-glycosylase gene (MPG), which functions in the ‘shortpatch-repair’ base-excision repair (BER) pathway (indicated by small arrows). Downregulation of MPG transcription might prevent the development of the mutator phenotype that might result from the increased accumulation of point mutations after NO-induced DNA damage. However, p53 can stimulate other components of the BER pathway, thereby providing a ‘delicate balancing act’ on BER activation. b | Examples of transactivation-independent functions of p53. The transactivation-independent functions of p53 can be exerted by different, yet complementary, mechanisms. p53 stimulates BER by interacting with APE1/REF1, 8-oxoguanine (8-oxoG) glycosylase (OGG1) and DNA polymerase β. p53 stimulates the activity of APE1/REF1 and OGG1. p53–DNA-polymerase-β interaction enhances the stability of the polymerase. In vivo, the APE1/REF1-mediated interaction of p53 with selenomethionine (SeMet) activates p53 and the DNA-repair machinery without affecting cell growth. In the presence of γ-irradiation or reactive oxygen species (ROS), p53 induces the activities of 3-MeAde DNA glycosylase or OGG1, respectively, which demonstrates the stress-dependent effect of p53 on BER enzymes.

(6-4) PHOTOPRODUCT

A type of DNA lesion that accounts for one quarter of all the DNA distortions and that are produced by moderate doses of UV irradiation. CHROMATIN-ACCESSIBILITY FACTOR

A factor that allows the detection and subsequent removal of bulky DNA adducts by ‘opening’ the chromatin. MICROSATELLITE INSTABILITY

A mutational change that occurs in the DNA, in which the number of repeats of microsatellites (short, repeated sequences of DNA) is different from the number of repeats that were in the DNA when it was inherited.

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multifunctional enzyme APE1/REF1, which has both endonuclease (APE1) and redox (REF1) activities. In vitro, p53 significantly enhances the excision of 8-oxoG nucleotides from the DNA by enhancing the sequential activities of OGG1 and APE1/REF1 (REF. 45). Interestingly, the level of DNA polymerase β is markedly diminished in p53-mutant or p53-null cells. This indicates the possibility that a DNA-polymeraseβ–p53 complex might affect the stability of the polymerase and that this stabilizing activity is absent in p53-deficient cells48. The integrity of the N-, core and C-terminal domains of the tumour suppressor are all required for its correct localization and the various interactions of p53 with the different components of the BER machinery 47. The question is still open whether p53-dependent transactivation is necessary for its role in BER. In vitro generated, transactivation-deficient, mutant p53 was shown to be more efficient in modulating BER activity than wild-type p53 (REF. 49). However, the same p53(22,23) mutant has also been proposed to lose the ability to stimulate BER due to the loss of DNA-polymerase-β interactions47 (FIG. 3b).


Selenomethionine (SeMet), which is the main source of selenium in our diet, provides yet another connection between p53 and APE1/REF1, as it regulates p53 by an APE1/REF1-dependent redox mechanism. Incubation with SeMet activates p53, and enhances its transactivation potential through changes in its conformation due to APE1/REF1-mediated reduction50. The DNA-binding activity of p53 is enhanced in the presence of wild-type APE1/REF1, but not its mutant form. The activated p53 becomes capable of enhancing the DNA-repair machinery without any growth-suppressive effects. As a result, the cells can tolerate higher doses of UV irradiation when grown in the presence of SeMet (FIG. 3b). Therefore, p53 can contribute to genomic stability by effectively inducing DNA repair, but not permanent growth arrest or apoptosis. Mismatch repair and p53 MICROSATELLITE INSTABILITY, which is characterized by mispaired nucleotides and insertions or deletions, is the consequence of slippage of the DNA polymerase during the synthesis of repetitive sequences in replication or recombination. Methylated bases of the type O6-methylguanine (O6MeG), paired with a C or a T, are also corrected by MMR due to the combined action of evolutionarily conserved repair-specific proteins. Among human germline mutations, the MMR proteins MutL homologue-1 (MLH1) and MutS homologue-2 (MSH2) together account for approximately half of all the hereditary non-polyposis colorectal cancer (HNPCC) patients12. However, recent evidence has indicated that the MMR system is not only involved in HNPCC, but in a much broader spectrum of human cancers51. MMR proteins and p53 have reciprocal effects on each other’s activity. The ataxia telangiectasia mutated (ATM)-mediated stabilization of the mismatch repair protein MLH1–postmeiotic-segregation-increased-2 (PMS2) heterodimer augments p53 activation during DNA damage52. This effect might have functional significance as male p53 –/– Msh2 –/– double-knockout mice show synergism in tumourigenesis53. So how does p53 affect MMR? p53 functions as a sequence-dependent transactivator by binding to several response elements in the promoter region of human MSH2 (REF. 54). In fact, p53 and the transcription factor Jun are reported to synergize in the regulation of human MSH2 in response to UV exposure. The human MSH2–MSH6 complex enhanced the in vitro binding of p53 to DNA substrates bearing bulged bases by 3–4-fold55, which indicates that MMR proteins and p53 might function synergistically in cells. MMR and p53 can cooperate to control the sensitivity to the cytotoxic effects of cisplatin and limit its mutagenic potential in colon cancer cells56. During S phase of the cell cycle, p53 and MSH2 bind efficiently to early recombination intermediates, they colocalize with each other and with the recombination proteins RAD50 and RAD51, and coexist within the same nuclear DNA–protein complexes. These results indicate that both MSH2 and p53 are recruited to the sites of recombination-associated repair and possibly modulate the process57.


REVIEWS The association between the lack of MSH2 or MLH1 expression in humans and the presence of p53 mutations has been observed in sarcoma and non-small-cell lung carcinoma58,59. Of clinical interest is the correlation between a p53 or MSH2 mutation and the diminished survival rate of hepatocellular carcinoma and head and neck cancer patients60,61. Microsatellite instability and p53 mutations are associated with the abnormal expression of the MSH2 gene in adult acute leukaemia62. The loss of p53 has relatively little effect on MMR-proficient cells, but confers substantial hypersensitivity to cisplastin on MMR-deficient cells56, which indicates that p53 and MMR cooperate in response to DNA damage. However, it has been reported that p53 and MMR mutations occur together in glioblastoma, but not in colorectal cancers63, which indicates a differential effect of p53 on the MMR system in diverse organs. Non-homologous end-joining and p53

V(D)J REARRANGEMENTS

The process of rearrangement and fusions of variable gene (V), diversity gene (D) and joining gene (J) segments that generate functional immunoglobin genes. HETERODUPLEX

A DNA duplex formed by the association between two homologous strands, each of which was previously hybridized to different complementary strands. If the homology is less than 100%, the heteroduplex will contain base mismatches that will require repair. HOLLIDAY JUNCTION

A cruciform DNA structure that is generated during the synaptic phase of homologous recombination.

NHEJ is the predominant DNA-repair process in G1 phase, but is also active in all other phases of the cell cycle64. In mammalian cells, most DNA double-strand breaks (DSBs) are repaired by NHEJ12,15. NHEJ is an ‘error-prone’ process as nucleotides at the break can be added or lost, and incorrect ends might be joined. However, NHEJ is still highly utilized to repair DSBs as, without it, large numbers of genetic alterations might accumulate. Limited inaccurate repair is tolerated in mammalian cells, given that a high percentage of the genome does not code for proteins. NHEJ is carried out by the combined action of different proteins, including DNA-PK (DNA-dependent protein kinase), XRCC4 (X-ray repair complementing defective repair in Chinese hamster cells-4) and DNA ligase IV (LIG4). DNA-PK consists of a catalytic subunit (DNA-PKcs) and a regulatory subunit Ku (a heterodimer of Ku70 and Ku80). DNA-PK phosphorylates and activates p53, which leads to apoptosis by transactivating downstream target genes65,66. However, other studies with a different strain of DNA-PKcs mice67 and a DNA-PK-defective cell line68 indicated that the p53mediated apoptotic response is intact, even in the absence of DNA-PK. p53 and DNA-PK form a complex in response to nucleoside analogues such as gemcitabine, and the complex interacts with gemcitabinecontaining DNA. The stalling of the DNA-PK–p53 complex at the site of drug incorporation stabilized and activated p53, and induced apoptosis69. Can p53 directly influence NHEJ, with or without the interaction with the specific repair proteins that control the process? The answer might be complicated. Both in vitro and in vivo studies show that wild-type p53 protein is capable of rejoining DNA with DSBs, which hints at its putative direct transactivation-independent role in NHEJ70,71. It has also been hypothesized that wild-type p53 can facilitate precise ligation72. Conversely, it has also been shown that DSB rejoining increases with the loss of wild-type p53 (REF. 73). Using reporter-gene reconstitution, integration or in vitro endjoining assays, wild-type p53 was reported to inhibit NHEJ74–77. In an attempt to bring these contradictory


results together, it was proposed that p53 inhibits errorprone, but not error-free, NHEJ 75. Analysis of double-knockout mice for individual NHEJ factors and p53 provide a fascinating look at the consequences of the failure to properly repair DSBs. Mice that are deficient in Ku, XRCC4 or LIG4 suffer extensive apoptosis of developing neurons, with Xrcc4 –/– and Lig4 –/– mice showing late embryonic lethality78–80, which is dramatically rescued by the homozygous deletion of p53 (REFS 81,82). However, the rescued doubleknockout Xrcc –/– p53 –/– and Lig4 –/– p53 –/– mice, as well as the Ku80 –/– p53 –/– mice, succumb to progenitor-B-cell lymphoma within 6–16 weeks after birth. The early death is due to the inability of the p53 deletion to rescue lymphocyte development in mice that lack NHEJ factors, which results in improper V(D)J REARRANGEMENTS and subsequent lymphomas bearing clonal and recurrent chromosomal rearrangements such as gene amplifications and translocations81–83. Therefore, the loss of NHEJ factors results in unrepaired DSBs and causes an apoptotic response by the p53-dependent activation, leading to embryonic lethality. Recent studies on doubleknockout (Artemis –/– p53 –/–) mice indicate that this NHEJ factor — Artemis (a nuclease that is mutated in a subset of human severe immunodeficient patients) — and p53 cooperate to suppress oncogenic N-Myc amplification84. So, Artemis –/– p53 –/– mice show amplification of N-Myc and succumb to progenitor-B-cell tumours. Homologous recombination and p53

HR is a fundamental and accurate process that is involved in maintaining the integrity of the genome and is conserved in all organisms. During HR, the sequence that is lost in one double-stranded-DNA molecule is replaced by the physical exchange of a segment from the second identical copy of the DNA12. After the detection of the DSB, the DNA is resected in the 5′→3′ direction and the resulting 3′ single-stranded tail is coated initially by RPA, which, in turn, stimulates the polymerization of the pro-recombinogenic protein, RAD51, and its subsequent interaction with DNA (this phase is referred to as the presynaptic phase; FIG. 4). RAD52 stimulates the polymerization of RAD51. The polymerized RAD51 searches the homology with the help of RAD54, a member of the SWI2/SNF2 (SWI refers to yeast mating-type switching, and SNF is an abbreviation for sucrose nonfermenting) family of helicases, which can bind to RAD51 stoichiometrically, stabilize the protein–DNA complex and thereby stimulate strand invasion85. Once the homology is located, the duplex is captured and the RAD51 filament invades the homologous duplex to form the HETERODUPLEX structure (synaptic phase). A DNA-dependent ATPase, RAD54, promotes this process by transiently denaturing the DNA base pairs. Then, heteroduplex-DNA extension and branch migration occurs, which constitutes the postsynaptic phase of the reaction. The intact double-stranded copy is used as a template for DNA synthesis by DNA polymerases. DNA ligases join the newly synthesized fragments, and the HOLLIDAY JUNCTIONS are resolved by specific endonucleases that are known as resolvases.


REVIEWS

DNA double-strand break

5′→3′ resection and coating with RPA

Strand invasion by polymerized RAD51

RAD54-promoted, Branch migration RAD51-mediated heteroduplex formation Heteroduplex

Resolution with resolvases; extension by DNA polymerase

Ligases

Product of homologous recombination

RPA Resolvase

RECQ HELICASE

A family of evolutionarily conserved helicases, mutations of which can lead to hereditary cancer-predisposition syndromes in humans.

50

RAD51

RAD52

p53

RAD54

Polymerase

Figure 4 | Role of p53 in homologous recombination. Homologous recombination (HR) is a multistep process that is mediated by the sequential accumulation of proteins. After detection of the DNA double-strand break (DSB) and resection of the DNA in the 5′→3′ direction, RAD51 binds to singlestranded (ss)DNA and displaces replication protein A (RPA), which leads to RAD51 polymerization (this phase is referred to as the presynaptic phase). Although RAD52 stimulates the polymerization of RAD51, wild-type p53 can control the process in vivo (the steps in the HR process that are controlled by p53 are shown) by its direct binding to RAD51. Once the homology search is successful, the duplex is captured and the RAD51 filament invades it to form the heteroduplex structure (synaptic phase). RAD54, a DNA-dependent ATPase, stabilizes the RAD51–ssDNA complex, thereby promoting this process. RAD54 can itself bind to p53 and scan the heteroduplex, a process that is probably regulated by p53 alone, or preferably targeted by RAD51. Depending on the extent of the damage, the mismatch is either corrected exonucleolytically by p53 or it can restrain the exchange of imperfectly homologous sequences. Heteroduplex DNA extension and branch migration normally occurs during the postsynaptic phase of HR, and wild-type p53 can inhibit the RAD51-promoted branch-migration process. The effect of p53 on heteroduplex or RAD51-mediated branch migration has been shown in in vitro studies. DNA polymerases use the intact copy to re-synthesize the deleted DNA sequences, DNA ligases join the newly synthesized fragments and the Holliday junctions are resolved by specific endonucleases that are known as resolvases.


p53 controls HR both in vitro and in vivo, and its inactivation results in enhanced spontaneous and stress-induced HR86–93. p53-deficient mice show an increased frequency of HR at different stages of development94. Furthermore, p53 with hotspot mutations at codons 281, 273, 248, 175 or 143 was found to be severely defective, with recombination frequencies that were elevated up to 26-fold75. This is probably because the cancer-derived p53 mutants (such as 273H) might not recognize the Holliday junctions95. In a cell line that co-expresses mutant p53, the deletion frequencies on assay plasmids were 5–20 times higher than in cells that expressed wild-type p53 alone90. The p53-dependent downregulation of HR is maximal between the sequences of short homologies75. Critical amino-acid residues that are required for p53-mediated transactivation have been identified96. Mutations in these residues lead to the generation of a transactivationdeficient mutant, p53(22,23). We and others have shown that p53 regulates both spontaneous and radiation-induced HR independently of its activities as a sequence-specific transcription factor and its subsequent involvement in G1–S checkpoint control89,93,97–99. The effect of p53 on HR can be either by itself or in association with HR-specific proteins, or a combination of both. This effect on HR can either be on the heteroduplex structure (as discussed in this section) or on Holliday junctions (in conjunction with RECQ HELICASES, as discussed in the next section). Wild-type p53 alone might check the fidelity of HR events by specific mismatch recognition in the heteroduplex intermediates86, and restrain DNA exchange between imperfectly homologous sequences, and so suppress tumourigenic genome rearrangements. The binding specificity of wild-type p53 to heteroduplex joint recombination intermediates depends on residues within the central DNA-binding core domain and the C-terminal tetramerization regions of p53. Tetramerized p53 stably binds to the strand-transfer regions, enabling the protein to exonucleolytically correct heteroduplex intermediates early after strand invasion95. p53 interacts with several proteins that are involved in the HR machinery, and one of the most studied is its interaction with and the control of RAD51. We have shown that the RAD51-mediated, elevated levels of HR that are seen in a host-cell reactivation assay can be controlled by a transactivation-deficient p53 (REF. 97,100). In vitro mapping studies indicate that RAD51 can bind to two regions of p53; one between amino acids 94 and 160, and a second between amino acids 264 and 315. The binding site of p53 on RAD51 between amino acids 125 and 220 is highly conserved between bacteria and humans and is necessary for polymerization101,102. In this context, it is pertinent to note that p53 also physically interacts with RAD54 and binds through its extreme C-terminal domain100 — the same region that also senses mispairings within HR intermediates89. Wild-type p53 (but not the tumour-derived mutant forms) abrogates wild-type RAD51 polymerization. Mutant RAD51 with reduced binding to p53 elevated HR and did not inhibit the polymerization, even in the presence of


REVIEWS

SISTER-CHROMATID EXCHANGE

An event that is similar to crossing over and that can occur between sister chromatids at mitosis or at meiosis. QUADRIRADIAL

A cytogenetic aberration involving symmetrical and asymmetrical interchanges between chromatids. CROSSING OVER

A reciprocal exchange of genetic information. TOPOISOMERASE

A class of enzymes that are involved in the regulation of DNA supercoiling. IONIZING RADIATION

Radiation, such as X-rays and γ-rays (high-energy photons), that causes atoms to release electrons and become ions. PML NUCLEAR BODY

(PML NB). One type of nuclear speckle that contains several proteins, including the promyelocytic leukaemia protein (PML). It is thought to be the site of recruitment of various proteins and might also have a role in gene transcription.

co-expressed wild-type p53 (REF. 100). The p53 (273H) mutant shows a reduced capacity to associate with RAD51–DNA complexes, even under conditions that support DNA binding102,103. These results indicate that p53 controls HR via direct interaction with and inhibition of RAD51 (FIG. 4). Apart from the controlling function of p53 on RAD51 during the presynaptic phase, the two proteins can also interact during the synaptic phase of HR. It has been proposed that p53 and RAD51 can interact during or shortly after strand transfer. In vitro, the p53–RAD51 complex can recognize the heteroduplex joints more efficiently than p53 alone, and RAD51 is involved in targeting p53 to these joints. RAD51 stimulates the inherent 3′→5′ exonuclease activity of p53, which p53 uses for the nucleolytic destruction of heteroduplexes that encompass base mispairings103. Apart from the synaptic phase, p53–RAD51 interaction might also function during the postsynaptic phase of HR. For example, it has been reported recently that, in vitro, wild-type p53, but not the p53 mutant proteins p53(248P) and p53(273P), inhibits branch migration promoted by RAD51 (REF. 104) (FIG. 4). It should be noted that many of the direct effects of RAD51 on p53 have been obtained from in vitro experiments. There is a degree of controversy over whether p53 can indeed act on extrachromosomal model systems, and it has been suggested that the regulation of HR by p53 is restricted to the highly ordered chromosomal chromatin structure105. Moreover, it has also been hypothesized that instead of inhibiting RAD51-mediated HR, p53 can protect mammalian cells from replication-associated DNA DSBs, possibly by suppressing the formation of recombinogenic lesions106. These issues need to be resolved by future experiments. Effect of p53 on RecQ helicases

RecQ helicases represent a subfamily of 3′→5′ DNA helicases that are highly conserved in evolution and are required for the maintenance of genomic integrity. Mutations in three of the RecQ-helicase genes in humans, BLM, WRN and RECQ4, lead to cancer-predisposition disorders — Bloom syndrome (BS), Werner syndrome (WS) and Rothmund–Thomson syndrome (RTS), respectively107. BS patient cells have enhanced rates of chromosomal instability and HR, as characterized by the elevated rates of SISTER-CHROMATID EXCHANGES, insertions, deletions, telomere associations and 107 QUADRIRADIALS . In vitro, recombinant BLM protein can modulate CROSSING-OVER events, along with TOPOISOMERASE III (REF. 108). WS cells also show aberrant HR. The defect is in the resolution stage of this process, which indicates that WRN is involved in the resolution of recombination structures109. The inter-regulation between p53 and the RecQ helicases has been shown at various levels. For example, the transcription factor Sp1-mediated expression of WRN is modulated by p53 at the transcription level. Mutation of Sp1-binding sites in the WRN promoter prevented repression by p53, which indicates that the p53–Sp1 complex might prevent Sp1 from


binding to the promoter110. At the post-transcriptional level, the C-terminal region of p53 interacts with either the N-terminal region of BLM or the C-terminal region of WRN in vitro111–114. In vivo, both BLM and WRN physically interact with p53 — possibly as part of a multiprotein complex that is involved in replication, recombination and repair97,112–115. A functional linkage between p53 and the RecQ helicases was obtained when it was reported that the ectopic co-expression of p53 with WRN in p53-deficient cells increases the level of transcriptionally active p53 levels. This corresponded with an enhancement in the level of the p53 downstream effector p21 and p53-mediated apoptosis114,116. During IONIZING RADIATION, p53-dependent apoptosis is attenuated in BS and WS cell lines and coincides with the physical interaction between p53 and BLM or WRN112,113. p53 can also affect the functions that are mediated by RecQ helicases. Wild-type p53, and not the mutant form, modulates the exonuclease activity of wild-type WRN, but not of a C-terminal-deletion mutant of WRN117. In addition to full-length, wild-type p53, a ten-amino-acid fragment of p53 (amino acids 373–383) can also attenuate the unwinding of Holliday junctions by BLM or WRN in vitro, whereas p53 phosphorylation at Ser376 and Ser378 completely abolishes this inhibition. Some of the cancer-derived p53 mutants reduce or abolish the ability of both WRN and BLM to bind to and unwind Holliday junctions115. This latter observation might have biological significance, because latent p53 is phosphorylated at Ser376 and is rapidly dephosphorylated by cellular stress118. Because the helicase function and nuclear localization of BLM or WRN are required for the restoration of stalled replication forks during replication stress97,117, it is perhaps not surprising that p53knockout mouse embryo fibroblasts show easily detectable DSBs during hydroxyurea (HU) treatment106. In vivo, p53 inactivation in BS cells causes a significant increase in sister-chromatid exchanges compared with BS cells that contain wild-type p53, thereby showing that p53 and BLM cooperatively affect HR and sisterchromatid exchanges97. The effect of p53 and BLM on HR is a direct consequence of complex intra-nuclear transport of these factors. BLM is an early sensor of HU-mediated replication stress. BLM and p53 colocalize and physically associate with each other and also with the HR factor RAD51. HU-mediated relocalization of BLM to RAD51 foci is p53 independent. However, BLM is required for efficient p53 accumulation to these sites and for the physical association of p53 with RAD51 (REF. 97). In non-stressed asynchronous cultures, BLM is found in 119,120 PML NUCLEAR BODIES (PML NBs) , which are regarded as the protein-storage depots of the nucleus. Cells that lack functional p53 accumulate lower amounts of BLM in PML NBs, whereas isogenic cell lines that have functional p53 show normal BLM accumulation under similar conditions113. These results could indicate that, whereas BLM might be necessary for the transport of p53 to sites of stalled DNA replication forks, p53 mediates nuclear trafficking of BLM back to the PML NBs.


REVIEWS

ANEUPLOIDY

The ploidy of a cell refers to the number of chromosome sets that it contains. Aneuploid karyotypes are chromosome complements that are not a simple multiple of the haploid set.

52

The HR process is influenced by a delicate balance between the pro-recombinogenic factors (for example, RAD51 and RAD54) and the anti-recombinogenic factors (such as p53 and BLM). The activity of the RAD51 recombinase during HR needs to be finely controlled as elevated levels of RAD51 are observed in various tumour cell lines, which indicates that hyper-recombination might have a role in tumourigenesis121,122. Therefore, there need to be checks and balances to allow the inherently accurate HR to proceed at optimum efficiency while preventing hyper-recombination. Several complexes among the anti-recombinogenic proteins, p53–BLM, p53–RAD51, p53–RAD54 and BLM–RAD54, have been described, which could possibly modulate and fine-tune the response and help to keep HR in balance97,100,107. Recent evidence from knockout mice reinforces the idea of a functional relationship between p53 and RecQ helicases. Wrn-deficient mice do not age prematurely in early generations123. However, Wrn –/– p53 –/– double-knockout mice show accelerated tumourigenesis and develop a broader spectrum of tumours than p53 –/– mice124. Moreover, the Wrn –/– p53 –/– mice show an accelerated mortality relative to Wrn +/– p53 –/– mice, which indicates that there are functional interactions between the p53 and the Wrn genes and their gene products. Recently, it has been reported that in late generations, mice that are doubly null for Wrn and the telomerase RNA component Terc have telomerase dysfunction, which elicits a classic WS premature-ageing-syndrome phenotype125. Crosses between the Wrn–/– Terc –/– and p53–/– mice might provide more answers regarding the interdependence of p53 and WRN during the ageing process. Uncontrolled hyper-recombination leads to genomic instability, including chromosomal deletions, translocations and gene amplifications. From the present literature, it is not obvious whether p53 loss alone can lead to hyper-recombination or whether p53 mutations, in conjunction with the loss of other downstream functions, lead to an increase in HR. In support of the sole role of p53, it has been shown in both genetic and cellular-knockout systems that the abrogation of wild-type p53 results in an increase in the frequency of spontaneous mutations and various chromosomal aberrations including deletions, amplifications and multiple translocations126–128. These results support the hypothesis that wild-type p53 alone is sufficient to facilitate recombinational DNA repair and so, the maintenance of genomic integrity. As an alternative to the above postulation, it has been reported that targeted inactivation of wild-type p53 by HR does not result in 129 ANEUPLOIDY . Increased rates of numerical or structural chromosomal instabilities were not observed in p53deficient cells. Rates of sister-chromatid exchange and HR were also unaffected by the p53 status in unstressed cells. So, it has been argued that p53 disruption alone is not sufficient to cause chromosomal instability. However, in this study only one single parental cancer cell line was used, and it is possible that this cell line could have carried changes in HR proteins, which could have masked the effects of changes in the p53 status.


p53: the ‘molecular node’

DNA-damage-induced cell-cycle checkpoints are signal-transduction cascades that link the detection of DNA damage to different downstream responses. There are three such gatekeepers in the eukaryotic cell cycle: the G1–S checkpoint (which inhibits the progression from G1 to S phase in the presence of DNA damage, thereby preventing the replication of damaged DNA), the intra-S-phase checkpoint (which is induced as a result of damage that occurred during S phase and to counter the lesions that had escaped the G1–S checkpoint) and the G2–M checkpoint (which prevents the progression of cells from G2 to M phase, thereby preventing mitosis in the presence of damaged DNA). Cells respond to the cell-cycle checkpoints either by transient growth arrest and the activation of DNA-damage-repair pathways or by the initiation of apoptosis. Although the stabilization of p53 is the most fundamental event in the G1–S checkpoint (see below), recent results indicate that the tumour suppressor also has important roles in the intra-S-phase checkpoint. The G1–S checkpoint is primarily induced by DSBs (FIG. 1). ATM represents the primary phosphoinositide3-kinase-like kinase (PIKK) responder to ionizingradiation-induced DSBs130. ATM phosphorylates key proteins in several signalling pathways either directly or indirectly through its in vivo substrate, checkpoint protein-2 (CHK2; REFS 130–132). Phosphorylated H2AX (γ-H2AX) marks the actual sites of DNA damage133. The phosphorylation of p53 can be mediated directly by ATM or indirectly via CHK2. So, one of the most important events in the G1–S checkpoint is the stabilization and activation of p53 (REFS 131,134). These results might be specific for normal human and mouse cells as in some tumour cells, CHK2 might lose its role as a stabilizing effector of p53 (REF. 135). The intra-S-phase checkpoint is triggered by replication stress that is caused by intrinsic events or by extrinsic genotoxic insults (for example, UV or HU) that prevent the progression of replication forks (FIG. 1). This pathway is primarily controlled by the ATR–ATRIP (ataxia-telangiectasia-and-RAD3 related–ATR-interacting-protein) complex136. ATR phosphorylates and activates CHK1, γ-H2AX137 and transducer proteins such as BLM138. We have recently shown that 53BP1 was required for the efficient accumulation of both BLM and p53 at the sites of stalled replication. The accumulation of BLM and p53 was independent of γ-H2AX. CHK1 phosphorylates BLM during replication stress and phosphorylated CHK1 was required for the accurate focal colocalization of 53BP1 with BLM, and the consequent stabilization of BLM 139. p53 can be activated by ATR and/or CHK1-mediated phosphorylation140,141 or indirectly via transducer proteins such as BLM, which transports p53 to the sites of stalled replication forks97. So, the signal during replication stress is transmitted through the ATR–CHK1–BLM–p53 pathway 97,138. This results in p53 stabilization and, consequently, the transcriptional attenuation of p53142.


REVIEWS So, p53 functions as a ‘molecular node’2 whereby the signals converge in response to either DSBs or stalled replication forks. p53 decides which effector role to assume, probably depending on the cell-cycle conditions and the type and extent of DNA damage. p53 can function either as a transactivator of genes that are involved in cell-cycle arrest, apoptosis or senescence, or as a modulator of DNA-repair and -recombination processes via either its transactivation-independent or -dependent functions. During the transactivationindependent process, p53 interacts with proteins that are involved in HR, NHEJ, MMR, BER and NER, thereby adding another aspect to its known role as a genome-surveillance protein (FIG. 1). p53: the cellular rheostat

The p53 response depends on its subcellular localization with respect to the site of DNA damage, the cellcycle status at the time of DNA damage and the dose and duration of the genotoxic stress. It can be envisaged that when the extent of DNA damage is low, the latent pool of p53, whether it is unmodified or posttranslationally modified, might interact with the DNA-repair machinery — either alone or in combination with other repair-specific factors. The advantage of such a graded response is that it prevents an ‘overreaction’ in response to the low level of DSBs and DNA distortions that might arise during the cell cycle or in response to low-level exposure to mediators of DNA damage. During the response to DNA damage, the upstream signalling process carries out a key role by recognizing the damage and post-translationally modifying p53. When the extent of DNA damage exceeds the level that can be successfully handled by p53 alone, the tumour suppressor undergoes

1.

Zhao, R. et al. Analysis of p53-regulated gene expression patterns using oligonucleotide arrays. Genes Dev. 14, 981–993 (2000). 2. Vogelstein, B., Lane, D. & Levine, A. J. Surfing the p53 network. Nature 408, 307–310 (2000). 3. Mirza, A. et al. Global transcriptional program of p53 target genes during the process of apoptosis and cell cycle progression. Oncogene 22, 3645–3654 (2003). 4. Polyak, K., Xia, Y., Zweier, J. L., Kinzler, K. W. & Vogelstein, B. A model for p53-induced apoptosis. Nature 389, 300–305 (1997). 5. Levine, A. J., Momand, J. & Finlay, C. A. The p53 tumour suppressor gene. Nature 351, 453–456 (1991). 6. Hollstein, M., Sidransky, D., Vogelstein, B. & Harris, C. C. p53 mutations in human cancers. Science 253, 49–53 (1991). 7. Ko, L. J. & Prives, C. p53: puzzle and paradigm. Genes Dev. 10, 1054–1072 (1996). 8. Liu, Y. & Kulesz-Martin, M. p53 protein at the hub of cellular DNA damage response pathways through sequencespecific and non-sequence-specific DNA binding. Carcinogenesis 22, 851–860 (2001). 9. Lee, S., Elenbaas, B., Levine, A. & Griffith, J. p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. Cell 81, 1013–1020 (1995). A key study that shows the in vitro binding of wild-type p53 to abnormal DNA structures. 10. Jiang, M. et al. p53 binds the nuclear matrix in normal cells: binding involves the proline-rich domain of p53 and increases following genotoxic stress. Oncogene 20, 5449–5458 (2001). 11. Aranda-Anzaldo, A., Orozco-Velasco, F., Garcia-Villa, E. & Gariglio, P. p53 is a rate-limiting factor in the repair of


stabilization, which is dependent on post-translational modification, and functions as a sequencedependent transcription factor. It thereby activates a set of genes that arrest the cell cycle, so that the DNArepair processes can successfully correct the lesion. During this step, p53 can also interact and modulate the different repair-process-specific proteins. If the DNA damage persists or is irreparable, apoptoticspecific genes are induced by p53, which leads to cell death. So, p53 serves as the ‘guardian of the genome’143 by functioning as the ‘cellular rheostat’ that modulates its multi-variant functions on the basis of the specific in vivo situation (FIG. 1). Although recent efforts have clarified the transactivation-independent functions of p53 during DNA repair and recombination, many questions remain. Mechanistically, it would be helpful to know whether and how p53 interacts with other, as-yet-unknown, components or auxiliary proteins that are involved in the different repair processes. Because p53 has an effect on DNA repair, replication and recombination, the multiprotein complexes are present at these sites and are coordinated by mutual interactions and p53 activity. The elucidation of the complexes in vivo, their hierarchy of importance, and the kinetics and dynamics of association with the DNA lesion should all be the subject of future research. Finally, the focus of this review has been to integrate the transactivation-independent functions of the tumour suppressor during DNA repair and recombination. Recent research has shown that the transactivation-independent function of p53 is also involved during apoptosis144. It could be imagined that there is mutual regulation between the different transactivation-independent functions of p53.

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Acknowledgments We thank J. Bradsher, X. Wang, J. Shen and S. Linke for helpful discussions. We also thank D. Dudek for editorial assistance and K. MacPherson for bibliographic assistance.

Competing interests statement The authors declare no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nlm.nih.gov/entrez/ BLM | RECQ4 | Terc | WRN OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM Bloom syndrome | HNPCC | Rothmund–Thomson syndrome | Werner syndrome | Xeroderma pigmentosum Swiss-Prot: http://www.expasy.org/ APE1 | ATM | ATR | ATRIP | CHK2 | CSA | CSB | DNA ligase IV | Jun | Ku70 | Ku80 | MDM2 | MLH1 | MSH2 | MSH6 | p48DDB2 | p53 | p127DDB1 | PCNA | PMS2 | RAD23B | RAD51 | RAD52 | Sp1 | XPA | XPB | XPC | XPD | XPF | XRCC4 Access to this links box is available online.
