Effect of wild-type, S15D and R175H p53 proteins on DNA end joining in vitro: potential mechanism of DNA double-strand break repair modulation

Andrei L. Okorokov,1, Lorna Warnock and Jo Milner

YCR p53 Research Group, Department of Biology, University of York, York, YO10 5DD, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Balanced regulation of DNA double-strand break (DSB) repair is crucial for genetic integrity and cell survival. Cells perform DSB repair either by homologous recombination (HR) or by non-homologous end joining (NHEJ). Either option carries risk for DNA instability. The presence in the cell of the tumour suppressor p53 has been shown to suppress the levels of HR; however, the effect of p53 on DNA EJ is less well understood. Here we demonstrate dramatically increased DNA EJ activity in cell-free extracts from p53–/– mouse embryo fibroblasts (MEFs) compared with p53+/+ MEFs. The addition of wild-type (wt) p53 to p53–/– MEFs extracts inhibited DNA EJ in a dose-dependent manner. Binding of wt p53 to DNA ends in vitro protected them from exonuclease attack and inhibited T4 DNA ligase-dependent EJ. This inhibitory effect was markedly enhanced for p53 R175H, a cancer-derived mutant of p53. In contrast, inhibition was negated in the presence of p53 S15D, a phosphorylation-mimicking mutant protein. Interestingly, p53 S15D stimulated in vitro DNA EJ of the blunt-ended DNA by T4 DNA ligase. Here we discuss the possibility that, in conjunction with its ability to control levels of HR, p53 may also serve to suppress DNA EJ in cells under normal conditions. This suppression may be associated with DNA-dependent protein kinases or ATM kinases, providing potential crosstalk between major cellular pathways of DNA repair and cell-cycle checkpoint mechanisms.

Abbreviations: DBS, double-strand breaks; DNA-PK, DNA-dependent protein kinase; HR, homologous recombination; NHEJ, non-homologous end joining


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recognition and repair of DNA double-strand breaks (DSBs) is essential for the maintenance of genetic integrity. Any event that interferes with this process results in genetic instability and, in higher eukaryotes, predisposes to cancer. Repair of DSBs can be roughly divided into two main pathways, namely: (i) homologous recombination (HR), when an undamaged sister-chromatid or homologous chromosome is used as a template for repair and (ii) non-homologous end joining (NHEJ) in which DNA ends are joined together with the use of little or no homology (reviewed in refs 1–4). Each repair option carries its own particular risk: EJ can be mutagenic at the site of the DSBs, whereas unregulated HR may lead to abnormal chromosomal rearrangements (5–7).

It is considered that a bias exists in vertebrates towards NHEJ as the dominant pathway for DSB repair. However, both pathways appear to distribute their functions preserving genomic integrity during the cell cycle. Thus, due to the availability of donor DNA template, HR is the preferential pathway during late S and G2 phases, whereas NHEJ is the primary DSB repair pathway in G0, G1 and early S phase of the cell cycle (8,9).

The major components of the HR pathway in mammals are the proteins of the Rad52 epistasis group, which include products of Rad50-55, Rad57, Rad59, MRE11 and NBS1 genes with Rad51 protein playing the central role. NHEJ is considered to be performed by cooperative action between the Ku protein heterodimer Ku70/Ku80 and catalytic subunit (DNA-PKcs) of DNA-dependent protein kinase (DNA-PK), XRCC4 protein and DNA ligase IV (LigIV) (10–12, reviewed in refs 1,3,4,7).

The importance of both pathways for viability and genomic integrity has been extensively demonstrated in studies on mice with genetic knockouts. Elimination of either HR- or NHEJ-related genes results in dramatic genomic instability and LigIV–/–, XRCC4–/– and Rad51–/– mice are inviable (13–18). A deficiency in DNA-PKcs, Ku70 or Ku80 does not lead to embryonic lethality; nevertheless, it results in impaired NHEJ, severe combined immunodeficiency, growth retardation, premature cellular senescence, chromosome breakage, massive translocations and aneuploidy (17,19–23).

The existing model proposes that the choice between HR and NHEJ when both pathways are available involves competition between Rad52 and Ku proteins. Thus, when Ku binds to the DSB it recruits XRCC4/DNA ligase IV heterodimer and channels repair into NHEJ (11,12,24) but, when Rad52 binds DSBs it initiates HR (2,25).

The process of DNA repair may be accompanied by stalling of the cell cycle and this can be achieved by the p53 tumour suppressor protein which is considered to be a classic `gatekeeper' of cellular fate (26). P53 is activated by diverse agents, which cause genotoxic stress. Once activated it initiates cell-cycle arrest via pathways involving the transactivation of p53 target genes. Alternatively, p53 induces apoptosis of the damaged cell, thus removing it from the healthy population.

Regarding DNA repair, p53 is mainly thought to provide an additional safeguard against genomic instability, which has been demonstrated by greatly increased chromosomal aberrations in Ku80–/–p53–/– mice (23). Loss of p53-dependent control can rescue animals with Rad51–/–, XRCC4–/– and LigIV–/– genotypes from early embryonic lethality but at the same time genomic instability dramatically increases (13,17,18,27).

An extensive body of in vivo observations indicates a close association between p53 and DNA repair. DNA DSBs are known to be an efficient inducer of p53 response within the cell (28,29). When cells derived from Li-Fraumeni Syndrome fibroblasts, which inherit one mutated (or deleted) allele of p53, become homozygous for mutant p53, this results in an extremely high frequency of gene amplification when compared with p53wt/mut or normal cells (30–32).

Similarly, human tumour cell lines, mutant or null for p53, show a large increase in spontaneous HR rates (33–35). Moreover, introduction of dominant negative mutants of p53 in mouse cells with wild-type (wt) p53 status resulted in a dramatic increase in spontaneous and radiation-induced intrachromosomal HR (36). Cells expressing mutant p53 have also been reported to exhibit high levels of deletions arising from HR. This prompted Gebow et al. (37) to suggest a model in which p53 may be directly involved in sensing DNA damage and co-ordinating the pathway(s) of repair.

Studies in vitro have demonstrated that p53 protein can bind to both double- and single-stranded DNA ends (38–40). Binding to DSBs can be regulated by a molecular switch mechanism with the potential of p53 serving as a `matchmaker' for DNA repair proteins (41). p53 possesses DNA re-annealing and 3' to 5' exonuclease activities (38,42). Human p53 has also been reported to bind to Holliday junctions and facilitate their cleavage by endonucleases (43). p53 has been found to bind insertion–deletion mismatches (39) and three-stranded DNA substrates, which mimic early recombination intermediates (44). Furthermore, p53 has been reported to interact with Rad51 protein and to inhibit Rad51 activity (45,46). Several p53 mutants were less capable of inhibiting Rad51, which suggested a possible molecular mechanism explaining the high rates of HR in p53 mutant cells (46).

A conflicting picture emerges from the literature on the role of p53 in DNA EJ. One report suggests that cell extracts from the transformed p53-deficient cells show increased levels of DNA EJ (47). In contrast, the presence of p53 has been reported to enhance EJ in irradiated transformed fibroblasts and thyroid cells (48–51).

Here we attempt to elucidate the role(s) of p53 in the modulation of DSB repair using an in vitro DNA EJ assay based on cell extracts from untransformed mouse embryo fibroblasts (MEFs) and purified p53 proteins. Our in vitro results suggest that wt p53 may suppress DNA EJ activity in vivo in non-stressed cells. This suppression appears to be regulated downstream by either DNA-PK or ATM kinases, providing potential crosstalk between major cellular pathways of DNA repair and cell-cycle checkpoint mechanisms.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasmid constructs and DNA substrates
Wt murine p53 cDNA was used as a template to produce p53 S15D, p53 S37D, p53 S15A, p53 S37A, p53 R175H, p53 M340Q/L344R and p53 50{Delta}C (residues 1–363) mutants as described previously (52). All numbering is for the corresponding amino acid residues in human p53. All cloned cDNAs were verified by DNA sequencing. For expression in the baculoviral system all target genes were subcloned into pVL1393 vector (PharMingen) and introduced into the baculoviral genome using BaculoGoldTM system (PharMingen).

For T4 DNA ligase and exonuclease assays, linear DNA substrate was prepared from pBluescript II SK(+) plasmid vector (Stratagene) purified by the `midi-prep' procedure (Qiagen) and linearized with EcoRI and EcoRV restriction enzymes (Promega) to generate 5'-cohesive and blunt ends, respectively. For DNA EJ in cell-free extracts, a linear DNA substrate was produced by PCR using the human p53 gene as a template and primers resulting in 1 kb PCR product and providing either EcoRI or EcoRV flanking sites at the ends. The DNA was uniformly 33P-labelled by addition of [33P]dATP (NEN Dupont) into the PCR reaction volume. PCR products were treated with either EcoRI or EcoRV restriction enzymes to generate 5'-protruding or blunt ends, respectively. Linearized DNA for all assays was finally purified using QIAquick spin columns (Qiagen) and DNA concentration was determined spectro-photometrically.

Recombinant proteins
Recombinant His-tagged human and murine wt p53 and murine p53 derivatives were expressed and purified to homogeneity as described previously (53). The final buffer used in the purification of p53 contained 50 mM NaC1, 5 mM MgC12, 10 mM Tris–HCl pH 7.0 and 5 mM DTT (`p53 buffer'). Protein concentration was quantified by the Bradford assay (Bio-Rad) and purity was checked by 15% SDS–PAGE followed by silver staining.

T4 DNA ligase assay
Linearized DNA was incubated with p53 (or its derivative) for 10 min at room temperature in 29 µl reaction mixture containing 10 µl of DNA (0.6 µg), 16 µl of p53 (amount is specified in the appropriate figure legend), 3 µl of 10x ligase buffer (300 mM Tris–HCl pH 7.8, 100 mM KC1, 100 mM DTT and 10 mM ATP). One hundred nanograms of tetrameric p53 protein mixed together with 0.5 µg of 3 kb linear DNA provided a protein to DNA end ratio of approximately 1:1. Subsequently, 1 µl of T4 DNA ligase (200 U, NEB) was added and the mixture incubated at 25°C for 1 or 2 h for 5'-cohesive or blunt-ended linear DNA, respectively. `p53 buffer' alone was used in control samples. Reactions were stopped and deproteinized by adding 3 µl of 2% SDS and 3 µl of 0.5 mg/ml Proteinase K (Boehringer) followed by 15 min incubation at 37°C. DNA ligation products were separated by 0.8% agarose gel electrophoresis in TAE buffer, visualized by staining with ethidium bromide and represented as an inverted image.

5'–3' exonuclease assay
EcoRI linearized pBluescript II SK(+) plasmid DNA (5'-cohesive ends) was pre-incubated with p53 protein for 15 min at room temperature in 39 µl reaction mixture containing 10 µl of DNA (0.5 µg), 25 µl of p53 (200 ng), -4 µl of 10x exonuclease buffer (660 mM Tris–HCl pH 8.0 and 6.6 mM MgC12). Subsequently, 1 µl (20 U) of exonuclease III (Promega) was added and the mixture incubated at room temperature for 5, 10, 20 or 30 min. `p53 buffer' alone was used in controls. Reactions were stopped and deproteinized by adding 5 µl of 0.5 M EDTA pH 8.0, 3 µl of 2% SDS and 3 µl of 0.5 mg/ml Proteinase K followed by 10 min incubation at 37°C. Products of the exonuclease treatment were separated by 0.8% agarose gel electrophoresis in TAE buffer, visualized by staining with ethidium bromide and represented as an inverted image.

DNA EJ in nuclear- and cell-free extracts
p53-/- and p53+/+ untransformed murine embryo fibroblasts were cultured in DMEM with 10% FCS (Gibco). Cells of seventh passage for both types of MEFs were used to prepare nuclear or total cell extracts. Nuclear extracts were prepared using NE-PERTM Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce) according to the manufacturers instructions. Total cell extracts were prepared essentially as described by Baumann and West (54). Cells were collected and washed three times in ice cold PBS and once in hypotonic lysis buffer (50 mM Tris–HCl pH 8.0, 10 mM NaCl, 1 mM EDTA and 5 mM DTT). Cells were resuspended in 2 vol of hypotonic buffer and, after 30 min on ice, were lysed by homogenization (20 strokes with `A' pestle) and protease inhibitors were added [1 mM AEBSF (Calbiochem), 5 mg/ml of aprotinin, 5 mg/ml of leupeptin, 10 µg/ml pepstatin A]. After 30 min on ice, 0.5 vol of high salt buffer (50 mM Tris–HCl -pH 7.5, 1 M KCl, 2 mM EDTA and 5 mM DTT) was added, and the extract was centrifuged for 3 h at 40 000 r.p.m. The supernatant was dialysed for 3 h against buffer containing 20 mM Tris–HCl pH 8.0, 0.1 M KOAc and 20% (vol/vol) glycerol, 1 mM AEBSF and 5 mM DTT, concentrated using Centricon 10 cartridges at +4°C, aliquoted (50 µl per every initial 106 cells), fast-frozen and stored in at –85°C. 33P-labelled linear DNA was incubated with p53 (or its derivative) for 10 min at room temperature in 40 µl reaction mixture containing 8 µl of DNA (0.6 µg), 25 µl of p53 (amount is specified in the appropriate figure legend), 2 µl of 0.1 M AEBSF and 5 µl of 10x ligase buffer (300 mM Tris–HCl pH 7.8, 100 mM KC1, 100 mM DTT and 10 mM ATP). Subsequently, 10 µl of total cell (or nuclear) extract (3.5 µg) were added and the mixture was incubated at 37°C for 2 h. `p53 buffer' alone was used in control samples. Reactions were stopped and deproteinized by adding 3 µl of 2% SDS and 3 µl of 0.5 mg/ml Proteinase K followed by 15 min incubation at 37°C. DNA EJ products were separated by 1% agarose gel electrophoresis in TAE buffer, vacuum dried at room temperature and analysed by autoradiography. Experiments with the DNA-PK inhibitor wortmannin (Sigma) or purified antibodies against Ku subunits Ku70 and Ku80 or p53 (Santa Cruz Biotechnology) were carried out by incubation of inhibitor or antibody with the extract for 10 min at 37°C before the addition of 33P-labelled linear DNA. Incubation then was continued for 1 h and terminated and analysed as above.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of wt p53 and p53 S15D on DNA EJ activity in p53–/– MEF cell-free extracts
Cell-free extracts were shown recently to be an adequate experimental system for the study of DNA EJ activity (54,55). We first analysed the difference in the levels of DNA EJ activity in cell-free extracts produced from MEFs originating from p53–/– and p53+/+ mice. The sensitivity of the assay was increased by using a linear DNA substrate, which was randomly labelled by the incorporation of 33P. DNA substrate was treated with EcoRI and EcoRV restriction enzymes to generate 5'-cohesive and blunt ends, respectively. Nuclear extracts (extract A) and total cell extracts (extract B) were prepared as described in Materials and methods.

Nuclear extracts from p53–/– MEFs showed considerable efficiency in DNA EJ of both 5'-cohesive and blunt-ended DNA resulting mainly in the formation of linear dimers with a limited amount of trimeric forms (Figure 1A and BGo, lanes 2 and 3). In contrast, the extracts obtained from p53+/+ MEFs failed to provide any higher rates of DNA EJ than background for both types of DNA target (Figure 1A and BGo, compare lanes 4 and 5 with control lane 1).




View larger version (76K):
[in this window]
[in a new window]
 
Fig. 1. Level of DNA EJ activity in p53–/– and p53+/+ MEF nuclear and total cell extracts. Non-transformed p53-/– and p53+/+ MEFs were used to prepare nuclear- or cell-free extracts. DNA EJ assays were performed as described in Materials and methods. 33P-labelled 1 kb linear DNA with either 5'-cohesive (A) or blunt ends (B) was used as a substrate. Nuclear extracts (extract A) from p53–/– MEFs show DNA EJ activity on both 5'-cohesive and blunt-ended DNAs (A and B, lanes 2 and 3). Nuclear extracts (extract A) obtained from p53+/+ MEFs show the same activity as background for both types of DNA target (A and B, compare lanes 4 and 5 with control lane 1). Similar differences can be observed for total cell extracts (extract B) from p53–/– and p53+/+ MEFs, compare lanes 6 and 7 (A and B) with lanes 8 and 9. Ten micrograms of total protein were used in lanes 2, 4, 6 and 8 and 30 µg of total protein were used in lanes 3, 5, 7 and 9. DNA EJ in nuclear extracts was inhibited in the presence of either wortmannin (C, lanes 3–5 compared with lane 2) or antibodies against Ku subunits (C, lanes 7–9). The control antibody (anti-p53) had no effect (C, lane 6). Control lanes (lane 1) represent samples of 5'-cohesive or blunt-ended linear DNA mixed with 30 µg of p53–/– MEF total cell extract and stop-mix was added immediately (see Materials and methods).

 
Total cell extracts from p53–/– MEFs showed higher efficiency than nuclear extracts resulting in the formation of linear di-, tri-, tetra- and higher oligomers in case of both types of DNA substrate (Figure 1A and BGo, lanes 7). This activity, however, was absent from cell-free extracts prepared from p53+/+ MEFs (Figure 1A and BGo, lanes 8 and 9).

The data indicate that the presence of wt p53 directly or indirectly leads to suppression of DNA EJ activity and that the loss of both p53 alleles results in increased DNA EJ activity. DNA EJ activity was decreased when DNA-PK inhibitor wortmannin, or anti-Ku70 and Ku80 antibodies were added to the reaction, indicating that most of the observed DNA EJ activity is Ku/DNA-PK-dependent (Figure 1CGo).

We next analysed the effect of the addition of wt p53 protein or its phosphorylation-mimicking mutant p53 S15D on the DNA EJ activity of total cell extracts obtained from untransformed p53–/– MEFs. Recombinant proteins were produced in the baculoviral system, purified to homogeneity and used in the cell-free DNA EJ assay (see Materials and methods).

The addition of increasing amounts of wt p53 protein resulted in a dose-dependent inhibition of the DNA EJ activity with both types of DNA substrate (Figure 2A and BGo, lanes 3 and 4 compared with lane 2). The addition of p53 S15D, however, failed to inhibit the DNA EJ process of both 5'-cohesive and blunt-ended DNA substrates in p53–/– MEF total cell extracts (Figure 2A and BGo, lanes 5 and 6).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2. p53 suppresses DNA EJ activity in p53–/– MEF total cell extracts. Comparison of DNA EJ products after 2 h at 37°C in the p53–/– MEFs cellular extract alone (30 µg of total protein, lane 1), or following pre-incubation with increasing amounts of wt p53 protein in 150 ng increments, from 150 to 300 ng (lanes 3 and 4) or p53 S15D (lanes 5 and 6). 5'-Cohesive (A) and blunt-ended (B) linear DNAs were 33P-labelled. Controls as described in Figure 1Go.

 
Effect of wt p53, p53 S15D and p53 S37D on EJ by T4 DNA ligase
We investigated the influence of p53 on the ligation of DSBs in vitro. For this purpose we used baculoviral-produced purified recombinant p53 proteins and T4 DNA ligase. pBluescript II SK(+) plasmid vector was the DNA substrate linearized with EcoRI or EcoRV to generate 5'-cohesive or blunt ends, respectively. Pre-incubation of the 5'-cohesive-ended linear DNA (3 kb arrowed) with the wt p53 protein resulted in decreased production of the supercoiled and high-oligomeric forms of DNA. The inhibitory effect was dose-dependent (Figure 3AGo, lanes 3–6, corresponding to wt). Similar results were obtained with purified human wt p53 (not shown).





View larger version (128K):
[in this window]
[in a new window]
 
Fig. 3. p53 affects DNA EJ by T4 DNA ligase. Comparison of ligation products of 5'-cohesive (A) and blunt-ended (B) linear DNA after 1 h at 25°C in the presence of 200 U T4 DNA ligase (NEB) alone (lane 2) or following pre-incubation with increasing amounts of wt p53 protein, p53 S15D or p53 S37D phosphorylation-mimicking mutants, and S15A non-phosphorylation mutant in 50 ng increments, from 100 to 250 ng (lanes 3–6). (C). Comparison of ligation products of 5'-cohesive (left panel) and blunt-ended (right panel) linear DNA after 1 h at 25°C in the presence of 200 U T4 DNA ligase (NEB) alone (lanes 2 and 9) or following pre-incubation with increasing amounts of wt p53 protein or with its dimeric and truncated p53 50{Delta}C mutants in 50 ng increments, from 100 to 250 ng (lanes 3–6 and 10–13). Lane 1 shows a control linear DNA (50% of the input) and lane 7 shows DNA size markers (Gene Ruler, MBI).

 
We also tested the effect of the phosphorylation-mimicking mutations at serine residues 15 and 37, on DNA-end ligation by T4 DNA ligase in vitro. Low amounts of p53 S15D displayed a similar effect on 5'-cohesive EJ to wt p53 (Figure 3Go, lanes 3 and 4, corresponding to S15D). However, on addition of higher protein amounts we observed higher oligomeric DNA forms (Figure 3AGo, lanes 5 and 6 corresponding to S15D).

The p53 S37D protein produced a less inhibitory effect on 5'-cohesive-ended DNA ligation than wt p53 (Figure 3AGo, lanes 3–6, corresponding to S37D). The results obtained with p53 S15A and S37A mutants as a control to the non-phosphorylated forms of p53 were essentially the same as those obtained with wt p53 (shown for S15A, Figure 3AGo, lanes 3–6).

When blunt-ended linear DNA was used as the substrate the activity of T4 DNA ligase was low, producing a small amount of dimeric forms of the linear DNA substrate (Figure 3BGo). The addition of wt p53 protein had a weak stimulatory effect (Figure 3BGo, lanes 3–5, corresponding to wt). Similar results were obtained with human wt p53 (not shown).

Surprisingly, the p53 S15D mutant showed dose-dependent stimulation of the EJ accompanied by increasing amounts of di-, tri- and higher oligomeric DNA forms (Figure 3BGo, lanes 3–5 corresponding to S15D). The effect produced by p53 S37D protein was not as profound as for p53 S15D (Figure 3BGo, corresponding to S37D). The properties of p53 S15A and S37A were similar to the wt p53 (shown for S15A, Figure 3BGo, lanes 3–5, corresponding to S15A).

We also tested the influence on ligation in vitro by dimeric p53 (p53 M340Q/L344R) and p53 truncated at the C-terminus (p53 50{Delta}C). Both dimeric and C-terminal truncated p53 proteins showed less inhibitory effect than wt p53 (Figure 3CGo). This was not unexpected as both modifications are predicted to decrease the stability of the complex between p53 and DNA ends. The monomeric central core domain of p53 (residues 98–323) had no inhibitory effect (data not shown).

p53 protects DNA ends against 5'–3' exonuclease attack
Interaction of p53 with DNA ends has been demonstrated previously by electron microscopy (39,40) and potentially explains the ability of p53 to modulate the kinetics of DNA EJ by T4 DNA ligase. If this were correct, the p53 protein would also be expected to protect linear DNA from exonuclease attack. Using 5'-cohesive-ended linear DNA substrate and exonuclease III (p53 has intrinsic 3'–5' exonuclease activity; 42) we have shown that in the absence of p53, the addition of exonuclease results in rapid degradation of DNA substrate over a 30 min time course (Figure 4Go, lanes 1–5). However, when DNA was pre-incubated with wt p53, the rate of degradation slowed down dramatically, with most of the initial DNA substrate remaining intact after 30 min (Figure 4Go, lanes 6–10).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4. p53 protects DNA ends against 5'–3' exonuclease attack. (A) 5'-Cohesive-ended linear DNA was incubated with 20 U of exonuclease III for 0, 5, 10, 20 and 30 min alone or following pre-incubation with 200 ng of wt p53, p53 S15D or p53 S37D mutant. (B) 5'-Cohesive-ended linear DNA was incubated with 20 U of exonuclease III for 0, 5, 20 and 30 min alone or following pre-incubation with 200 ng of wt p53, dimeric or truncated p53 50{Delta}C mutant.

 
We also compared the ability of S15D and S37D phosphorylation-mimicking mutants to protect 5'-cohesive DNA ends against exonuclease III. Both mutants demonstrated end-protection properties similar to those of wt p53 (Figure 4Go, lanes 11–15 and 16–20, corresponding to S15D and S37D, respectively). Similar results were obtained with p53 S15A and S37A mutants and when a range of concentrations of p53 proteins was used in the assay (not shown). These results indicate that the difference in the inhibitory effect of these mutants of p53 on DNA EJ is not due to the differential DNA binding abilities between wt p53 and its phosphorylation-mimicking mutants.

However, when we tested dimeric and C-terminal truncated p53 proteins they exerted less protection than wt p53 (Figure 4BGo). P53 50{Delta}C protein showed a lower efficiency in protection against exonuclease attack, with only some linear DNA remaining intact after 30 min of incubation (Figure 4BGo compare lanes 8, 12 and 16). No effects were observed when p53 (98–323) central core domain has been used as a control (not shown).

Cancer-derived p53 R175H mutant suppresses DNA EJ in vitro more efficiently than wt p53
We then tested the effect of p53 mutation at codon 175, one of the most commonly altered codons in human cancer that also linked to dramatically raised levels of chromosomal aberrations. Here we used the site-directed engineered mutant p53 R175H, produced and purified from the baculoviral system. We first tested the effect of p53 R175H on T4 DNA ligase-dependent DNA EJ. The R175H mutant showed a remarkably stronger inhibitory effect on ligation of 5'-ended linear DNA than wt p53 (Figure 5AGo, compare lanes 3 and 4).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 5. p53 R175H mutant affects DNA EJ in vitro more efficiently than wt p53. (A) Comparison of ligation products of 5'-cohesive-ended linear DNAs after 1 h in the presence of 200 U of T4 DNA ligase (NEB) alone (lane 2) or following pre-incubation with 100 ng of wt p53 protein (lane 3) or the p53 R175H mutant (lane 4). Lane 1 shows control linear DNA and lane 5 shows DNA size markers (Gene Ruler, MBI). (B) Comparison of ligation products of 5'-cohesive-ended linear DNAs after 1 h in the presence of 200 U of T4 DNA ligase (NEB) alone (lane 2) or following pre-incubation with increasing amounts of wt p53 protein or the p53 R175H proteins in 50 ng increments from 100 to 250 ng (lanes 3–6). Lane 1 shows control linear DNA and lane 7 shows DNA size markers (Gene Ruler, MBI). (C) Comparison of DNA EJ products after 2 h at 37°C in the p53–/– MEFs cellular extract alone (30 µg of total protein, lane 2), or following pre-incubation with increasing amounts of wt p53 protein in 100 ng increments, from 100 to 200 ng (lanes 3 and 4) or p53 R175H (lanes 5 and 6). 5'-Cohesive-ended linear DNA was 33P-labelled. Control as described in Figure 1Go.

 
Titration of different amounts of wt p53 and p53 R175H proteins showed that this structural mutant inhibits DNA ligase activity ~10-fold stronger than wt p53 (Figure 5BGo). This was not, however, due to a higher efficiency of p53 R175H binding to the DNA ends, as the exonuclease protection assay did not show any difference between R175H mutant and wt p53 proteins (not shown).

We also looked at the inhibitory effect of p53 R175H mutant protein on DNA EJ in p53–/– MEFs total cell extracts. The addition of low concentrations of wt p53 protein demonstrated a slight inhibitory effect on DNA EJ (Figure 5CGo, lane 4). The addition of similarly low amounts of p53 R175H mutant protein to the total cell extracts produced a greater inhibitory effect than wt p53 (Figure 5CGo, lanes 5 and 6). Similar results were obtained when blunt-ended linear DNA was used as a substrate (not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been reported previously that cell extracts from p53-deficient transformed cells show increased levels of DNA EJ (47). Furthermore, transformed p53mut cells were more proficient at the re-joining of DNA DSBs after irradiation than cells with wt p53 (49,50). Mallya and Sikpi (50) suggested that p53 may participate in suppressing DSB re-joining following exposure of mammalian cells to irradiation. In contrast, the presence of wt p53 has been reported to enhance DNA EJ in irradiated cells when compared with p53null cells (48,51). Thus, irradiated transformed rat thyroid cells exhibit higher EJ activity after low-dose radiation (48). Similarly, wt p53-dependent higher efficiency of DNA end re-joining was observed in irradiated untransformed MEFs (51).

To address the conflicting data and to avoid limitations of in vivo experimental systems when addressing the role(s) of p53 in the modulation of DNA repair, we attempted to study the potential role(s) of p53 in the modulation of DNA EJ using an in vitro DNA EJ assay. We demonstrate that nuclear and total cell extracts derived from untransformed p53+/+ MEFs show no detectable DNA EJ activity whereas similar extracts from untransformed p53–/– MEFs exhibit considerable DNA EJ activity with either 5'- or blunt-ended DNA (Figure 1Go). Similar findings were reported for primary cells using an in vivo plasmid assay (51); thus, our in vitro assay is consistent with in vivo experimental approaches.

We also demonstrate that the addition of the purified tumour suppressor protein p53 inhibits the DNA EJ activity in total cell extracts prepared from untransformed p53–/– MEFs (Figure 2Go).

The multifunctional protein, p53, may participate in the modulation of spontaneous DNA EJ both directly and indirectly. This may explain the extremely low DNA EJ activity levels in p53+/+ MEF extracts. One may expect a difference in the DSB repair activity in MEFs with p53–/– or p53+/+ genotypes due to their difference in rate of growth. However, no alterations in DSB repair protein levels were reported for cell lines established from human fibroblasts with varying radiosensitivities and moreover, changes in repair activity did not correlate with cellular radiosensitivity (56).

One may suggest that this observed suppression of DNA EJ activity in cell extracts by wt p53 follows on from the ability of p53 to bind DNA ends and, therefore, to compete with NHEJ proteins. When tested in vitro, wt p53 affects the activity of T4 DNA ligase on 5'-cohesive ends DNA ends and to a lesser extent DNA blunt ends (Figure 3Go). p53 also provides protection of DNA ends against exonuclease treatment (Figure 4Go). The situation in vivo, however, may be different as DNA ligase and/or exonucleases will presumably compete with additional NHEJ components.

This activity depends on the stability of the p53/DNA-end complex as p53 derivatives such as p53 50{Delta}C and dimeric p53 which form less stable complexes, both show reduced inhibition of T4 DNA ligase and exonuclease III activity (Figures 3 and 4GoGo).

Protein–protein interaction between (or within) the p53 molecules at DNA ends may also play an important role in p53s modulation of DSB repair. Our data indicate that although wt p53 and its phosphorylation-mimicking mutants all have similar efficiency in binding DNA ends and protecting them against 5'–3' exonuclease treatment, they show marked differences in DNA EJ suppression. The difference between p53 S15D and S15A, S37D and S37A mutants also provides an internal control in that not every modification within the N-terminus of p53 has a similar effect on DNA EJ.

Serine 15 is the target site of DNA-PK and ATM kinases, which are both involved in DNA damage-induced cellular responses (57–60). p53 was found to be phosphorylated at position Ser15 in vivo in response to {gamma}-irradiation and this is considered to be a part of the activation and stabilization processes of p53 (61–63, reviewed in ref. 26). Our data show that the addition of p53 S15D protein to the p53–/– MEFs total cell extracts had no inhibitory effect on DNA EJ (Figure 2Go). Moreover, this phosphorylation-mimicking mutant stimulated the DNA EJ by T4 DNA ligase of the blunt-ended DNA linear substrate (Figure 3BGo) and may be a direct participant in DNA repair. It would be plausible to suggest that placing the negative charge at the residue Ser15 modulates the properties of p53 in respect of its intermolecular interaction. However, bacterial DNA ligases have markedly different properties from eukaryotic nuclear DNA ligases; therefore, we are unable to extrapolate further from our finding that p53 S15D stimulates EJ by T4 DNA ligase.

It has been shown, that most DSBs in the cell are repaired within the time which precedes detectable p53 accumulation, leaving p53 outplayed by Ku in terms of numbers of molecules (64). Therefore, a physiological situation can be imagined in which the potential role of p53 is to receive the damage signal from DNA repair machinery rather than directly to participate in the DNA repair process.

DNA-PKcs is known to phosphorylate Ku, LigIV and XRCC4 proteins and in trans, itself (57,65–68). These phosphorylation events are thought to be part of (i) stimulation of activity of LigIV and XRCC4 and (ii) necessary for the disassembly of the complex (reviewed in ref. 60). Auto-phosphorylation of DNA-PKcs on the other hand inactivates its own kinase activity, probably serving as a feedback mechanism (65). The phosphorylation of p53 by DNA-PK in vivo is most probably performed by DNA-PKcs at the site of DSBs, where it exerts kinase activity. Interaction of p53 with DNA-PK potentially followed by phosphorylation may serve to disassemble the p53–DSB complex. At the same time, this interaction may trigger a signalling mechanism for p53 phosphorylation/activation. We suggest that phosphorylation of p53 at Ser15 may be required to relieve the p53-dependent suppression of DNA EJ and/or for the disassembly of p53 from the protein machinery recruited to the DNA end.

The molecular basis for the p53-dependent control of HR was proposed to be the suppression of Rad51 activity and indeed, structural mutants of p53 were reported to be far less efficient in inhibiting Rad51 (45,46). Other studies suggest that p53 may also play a role in the fidelity of the HR machinery, causing proofreading deficiency in the presence of a p53 mutation (37,44,69). We demonstrate here that the R175H p53 mutant protein has also an increased ability to suppress DNA EJ in p53–/– MEF cell-free extracts compared with wt p53 (Figure 4Go).

We suggest that some structural mutants of p53 may have a profound dominant negative effect on chromosomal integrity not only due to the loss of control of HR and the inability to initiate the cell-cycle checkpoint but also due to their influence on the DNA EJ cellular response to spontaneous DNA DSBs. In addition, mutant p53 may not be able to perform its role as a transcription activation factor, up regulating the expression of proteins involved directly or contributing to the fidelity of DNA DSB repair.

Overall, our data indicate that p53 may serve to suppress spontaneous DNA EJ in the cell under normal conditions. This suppression may be associated with DNA-PK or ATM kinases, providing potential additional crosstalk between major cellular pathways of DNA repair and cell-cycle checkpoint mechanisms. House-keeping error-free DNA EJ repair machinery may be affected by mutant p53, giving way to either error-prone NHEJ or spontaneous HR, no longer under p53-dependent control. A failure in cell-cycle checkpoint activation would guarantee that chromosomal aberrations were generated in subsequent cell cycles.


    Notes
 
1 To whom correspondence should be addressed Email: a.okorokov{at}mail.cryst.bbk.ac.uk Back


    Acknowledgments
 
We thank Guillermina Lozano for the kind gift of p53–/– and p53+/+ MEFs. This work was supported by a Yorkshire Cancer Research program grant to J.M.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Critchlow,S.E and Jackson,S.P. (1998) DNA end-joining: from yeast to man. Trends Biochem. Sci., 23, 394–398.[ISI][Medline]
  2. Haber,J.E. (1999) Gatekeepers of recombination. Nature, 398, 665–667.[ISI][Medline]
  3. Dasika,G.P., Lin,S.C., Zhao,S., Sung,P., Tomkinson,A. and Lee,E.Y.H.P. (1999) DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis. Oncogene, 18, 7883–7899.[ISI][Medline]
  4. Karran,P. (2000) DNA double strand break repair in mammalian cells. Curr. Opin. Genet. Dev., 10, 144–150.[ISI][Medline]
  5. Derbyshire,M.K., Epstein,L.H., Young,C.S.H., Munz,P.L. and Fishel,R. (1994) Nonhomologous recombination in human cells. Mol. Cell. Biol., 14, 156–169.[Abstract]
  6. Friedberg,E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and Mutagenesis. American Society for Microbiology, Washington, DC.
  7. Baumann,P. and West,S.C. (1998) Role of the human RAD51 protein in homologous recombination and double-stranded-break repair. Trends Biochem. Sci., 23, 247–251.[ISI][Medline]
  8. Takata,M., Sasaki,M.S., Sonoda,E., Morrison,C., Hashimoto,M., Utsumi,H., Yamaguchi-Iwai,Y., Shinohara,A. and Takeda,S. (1998) Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J., 17, 5497–5508.[Abstract/Free Full Text]
  9. Essers,J., van Steeg,H., de Wit,J., Swagemakers,S.M.A., Vermeij,M., Hoeijmakers,J.H.J. and Kanaar,R. (2000) Homologous and non-homologous recombination differentially affect DNA damage repair in mice. EMBO J., 19, 1703–1710.[Abstract/Free Full Text]
  10. Lee,S.E., Mitchell,R.A., Cheng,A. and Hendrickson,E.A. (1997). Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle. Mol. Cell. Biol., 17, 1425–1433.[Abstract]
  11. Ramsden,D.A. and Gellert,M. (1998) Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks. EMBO J., 17, 609–614.[Abstract/Free Full Text]
  12. McElhinny,S.A., Snowden,C.M., McCarville,J. and Ramsden,D.A. (2000) Ku recruits the XRCC4-ligase IV complex to DNA ends. Mol. Cell. Biol., 20, 2996–3003.[Abstract/Free Full Text]
  13. Lim,D.S. and Hasty, P. (1996) A mutation in mouse rad51 results in an early embryonic lethality that is suppressed by a mutation in p53. Mol. Cell. Biol., 16, 7133–7143.[Abstract]
  14. Tsuzuki,T., Fujii,Y., Sakumi,K., Tominaga,Y., Nakao,K., Sekiguchi,M., Matsushiro,A., Yoshimura,Y. and Morita,T. (1996) Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl Acad. Sci. USA, 93, 6236–6240.[Abstract/Free Full Text]
  15. Grawunder,U., Wilm,M., Wu,X., Kulesza,P., Wilson,T.E., Mann,M. and Lieber,M.R. (1997) Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature, 388, 492–495.[ISI][Medline]
  16. Grawunder,U., Zimmer,D., Fugmann,S., Schwarz,K. and Lieber,M.R. (1998) DNA ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes. Mol. Cell, 2, 477–484.[ISI][Medline]
  17. Ferguson,D.O., Sekiguchi,J.M., Chang,S., Frank,K.M., Gao,Y., DePinho,R.A. and Alt,F.W. (2000) The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations. Proc. Natl Acad. Sci. USA, 97, 6630–6633.[Abstract/Free Full Text]
  18. Frank,K.M., Sharpless,N.E., Gao,Y., Sekiguchi,J.M., Ferguson,D.O., Zhu,C., Manis,J.P., Horner,J., DePinho,R.A. and Alt,F.W. (2000) DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway. Mol. Cell, 5, 993–1002.[ISI][Medline]
  19. Zhu,C., Bogue,M.A., Lim,D.S., Hasty,P., and Roth,D.B. (1996) Ku80-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell, 86, 379–389.[ISI][Medline]
  20. Gu,Y., Jin,S., Gao,Y., Weaver,D.T. and Alt,F.W. (1997) Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity and inability to support V(D)J recombination. Proc. Natl Acad. Sci. USA, 94, 8076–8081.[Abstract/Free Full Text]
  21. Karanjawala,Z.E., Grawunder,U., Hsieh,C.L. and Liber,M.R. (1999) The nonhomologous DNA end-joining pathway is important for chromosome stability in primary fibroblasts. Curr. Biol., 9, 1501–1504.[ISI][Medline]
  22. Lim,D.S., Vogel,H., Willerford,D.M., Sands,A.T., Platt,K.A. and Hasty,P. (2000) Analysis of ku80-mutant mice and cells with deficient levels of p53. Mol. Cell. Biol., 20, 3772–3780.[Abstract/Free Full Text]
  23. Difilippantonio,M.J., Zhu,J., Chen,H.T., Meffre,E., Nussenzweig,M.C., Max,E.E., Ried,T. and Nussenzweig,A. (2000) DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature, 404, 510–514.[ISI][Medline]
  24. Jin,S. and Weaver,D.T. (1997) Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions. EMBO J., 17, 6874–6885.
  25. Van Dyck,E., Stasiak,A.Z. and West,S.C. (1999) Binding of double-stranded breaks in DNA by human Rad52 protein. Nature, 398, 728–731.[ISI][Medline]
  26. May,P. and May,E. (1999) Twenty years of p53 research: structural and functional aspects of the p53 protein. Oncogene, 18, 7621–7636.[ISI][Medline]
  27. Gao,Y, Ferguson,D.O, Xie,W., Manis,J.P., Sekiguchi,J., Frank,K.M., Chaudhuri,J., Horner,J., DePinho,R.A. and Alt,F.W. (2000) Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development. Nature, 404, 897–900.[ISI][Medline]
  28. Nelson,W.G. and Kastan,M.B. (1994) DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol. Cell. Biol., 14, 1815–1823.[Abstract]
  29. Huang,L.C., Clarkin,K.C. and Wahl,G.M. (1996) Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest. Proc. Natl Acad. Sci. USA, 93, 4827–4832[Abstract/Free Full Text]
  30. Livingstone,L.R., White,A., Sprouse,J., Livanos,E., Jacks,T. and Tlsty,T.D. (1992) Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell, 70, 923–935.[ISI][Medline]
  31. Yin,Y., Tainsky,M.A., Bischoff,F.Z., Strong,L.C. and Wahl,G.M. (1992) Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell, 70, 937–948.[ISI][Medline]
  32. Malkin,D. (1994) P53 and the Li-Fraumeni syndrome. Biochim. Biophys. Acta Rev. Cancer, 1198, 197–213.[ISI][Medline]
  33. Mekeel,K.L., Tang,W., Kachnic,L.A., Luo,C.M., DeFrank,J.S. and Powell,S.N. (1997) Inactivation of p53 results in high rates of homologous recombination. Oncogene, 14, 1847–1857.[ISI][Medline]
  34. Honma,M., Zhang,L.S., Hayashi,M., Takeshita,K., Nakagawa,Y., Tanaka,N. and Sofuni,T. (1997) Illegitimate recombination leading to allelic loss and unbalanced translocation in p53-mutated human lymphoblastoid cells. Mol. Cell. Biol., 17, 4774–4781.[Abstract]
  35. Bertrand,P., Rouillard,D., Boulet,A., Levalois,C., Soussi,T. and Lopez,B.S. (1997) Increase of spontaneous intrachromosomal homologous recombination in mammalian cells expressing a mutant p53 protein. Oncogene, 14, 1117–1122.[ISI][Medline]
  36. Saintigny,Y., Rouillard,D., Chaput,B., Soussi,T. and Lopez,B.S. (1999) Mutant p53 proteins stimulate spontaneous and radiation-induced intrachromosomal homologous recombination independently of the alteration of the transactivation activity and of the G1 checkpoint. Oncogene, 18, 3553–3563.[ISI][Medline]
  37. Gebow,D., Miselis,N. and Liber,H.L. (2000). Homologous and nonhomologous recombination resulting in deletion: effects of p53 status, microhomology and repetitive DNA length and orientation. Mol. Cell. Biol., 20, 4028–4035.[Abstract/Free Full Text]
  38. Oberosler,P., Hloch,P., Ramsperger,U. and Stahl,H. (1993) p53-catalyzed annealing of complementary single-stranded nucleic acids. EMBO J., 12, 2389–2396.[Abstract]
  39. Lee,S., Elenbaas,B., Levine,A. and Griffith,J. (1995) p53 and its 14 kDa C-terminal domain recognize primary DNA-damage in the form of insertion/deletion mismatches. Cell, 81, 1013–1020.[ISI][Medline]
  40. Bakalkin,G., Selivanova,G., Yakovleva,T., Kiseleva,E., Kashuba,E., Magnusson,K., Szekely,L., Klein,G., Terenius,L. and Wiman,K. (1995) p53 binds single-stranded DNA ends through the C-terminal domain and internal DNA segments via the middle domain. Nucleic Acid Res., 23, 362–369.[Abstract]
  41. Okorokov,A.L. and Milner,J. (1999) ATP/ADP-dependent molecular switch regulates the stability of p53-DNA complexes. Mol. Cell Biol., 19, 7501–7510.[Abstract/Free Full Text]
  42. Mummenbrauer,T., Janus,F., Mueller,B., Wiesmueller,L., Deppert,W. and Grosse,F. (1996) p53 protein exibits 3'- to 5'-cohesive exonuclease activity. Cell, 85, 1089–1099.[ISI][Medline]
  43. Lee,S., Cavallo,L. and Griffith,J. (1997) Human p53 binds Holliday junctions strongly and facilitates their cleavage. J. Biol. Chem., 272, 7532–7539.[Abstract/Free Full Text]
  44. Dudenhoffer,C., Rohaly,G., Will,K., Deppert,W. and Wiesmuller,L. (1998) Specific mismatch recognition in heteroduplex intermediates by p53 suggests a role in fidelity control of homologous recombination. Mol. Cell. Biol., 18, 5332–5342.[Abstract/Free Full Text]
  45. Sturzbecher,H.W., Donzelmann,B., Henning,W., Knippschild,U. and Buchhop,S. (1996) P53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. EMBO J., 15, 1992–2002.[Abstract]
  46. Buchhop,S., Gibson,M.K., Wang,X.W., Wagner,P., Sturzbecher,H.W. and Harris,C.C. (1997) Interaction of p53 with the human Rad51 protein. Nucleic Acids Res., 25, 3868–3874.[Abstract/Free Full Text]
  47. Bill,C.A., Yu,Y., Miselis,N.R., Little,J.B. and Nickoloff,J.A. (1997) A role for p53 in DNA end rejoining by human cell extracts. Mutat. Res., 385, 21–29.[ISI][Medline]
  48. Yang,T., Namba,H., Hara,T., Takmura,N., Nagayama,Y., Fukata,S., Ishikawa,N., Kuma,K., Ito,K. and Yamashita,S. (1997) p53 induced by ionizing radiation mediates DNA end-jointing activity, but not apoptosis of thyroid cells. Oncogene, 14, 1511–1519.[ISI][Medline]
  49. Mallya,S.M. and Sikpi,M.O. (1998) Evidence of the involvement of p53 in gamma-radiation-induced DNA repair in human lymphoblasts. Int. J. Radiat. Biol., 74, 231–8[ISI][Medline]
  50. Mallya,S.M. and Sikpi,M.O. (1999) Requirement for p53 in ionizing-radiation-inhibition of double-strand-break rejoining by human lymphoblasts. Mutat. Res., 434, 119–32.[ISI][Medline]
  51. Tang,W., Willers,H. and Powell,S.N. (1999) p53 directly enhances rejoining of DNA double-strand breaks with cohesive ends in gamma-irradiated mouse fibroblasts. Cancer Res., 59, 2562–2565.[Abstract/Free Full Text]
  52. Okorokov,A.L., Ponchel,F. and Milner,J. (1997) Induced N- and C-terminal cleavage of p53: a core fragment of p53, generated by interaction with damaged DNA, promotes cleavage of the N-terminus of full-length p53, whereas ssDNA induces C-terminal cleavage of p53. EMBO J., 16, 6008–6017.[Abstract/Free Full Text]
  53. Mee,T., Okorokov,A.L., Metcalfe,S. and Milner, J. (1999) Proteolytic cleavage of p53 mutants in response to mismatched DNA. Br. J. Cancer, 81, 212–218.[ISI][Medline]
  54. Baumann,P. and West,S.C. (1998) DNA end-joining catalyzed by human cell-free extracts. Proc. Natl Acad. Sci. USA, 95, 14066–14070.[Abstract/Free Full Text]
  55. Labhart,P. (1999) Nonhomologous DNA end joining in cell-free systems. Eur. J. Biochem., 265, 849–861.[Abstract/Free Full Text]
  56. Carlomagno F. et al. (2000) Comparison of DNA repair protein expression and activities between human fibroblast cell lines with different radiosensitivities. Int. J. Cancer, 85, 845–849.[ISI][Medline]
  57. Lees-Miller,S.P., Sakaguchi,K., Ullrich,S.J., Appella,E. and Anderson,C.W. (1992) Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol. Cell. Biol., 12, 5041–5049.[Abstract]
  58. Kim,S.T., Lim,D.S., Canman,C.E. and Kastan,M.B. (1999) Substrate specificities and identification of putative substrates of ATM kinase family members. J. Biol. Chem., 274, 37538–37543.[Abstract/Free Full Text]
  59. Rotman,G. and Shiloh,Y. (1999) ATM: a mediator of multiple responses to genotoxic stress. Oncogene, 18, 6135–6144.[ISI][Medline]
  60. Smith,G.C. and Jackson,S.P. (1999) The DNA-dependent protein kinase. Genes Dev., 13, 916–934.[Free Full Text]
  61. Shieh,S.Y., Ikeda,M., Taya,Y. and Prives,C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell, 91, 325–334.[ISI][Medline]
  62. Siliciano,J.D., Canman,C.E., Taya,Y., Sakaguchi,K., Appella,E. and Kastan,M.B. (1997) DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev., 11, 3471–3481.[Abstract/Free Full Text]
  63. Tibbets,R.S., Brumbaugh,K.M., Williams,J.M., Sarkaria,J.N., Cliby,W.A., Shieh,S.Y., Taya,Y., Prives,C. and Abraham,R.T. (1999) A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev., 13, 152–157.[Abstract/Free Full Text]
  64. DiBiase,S.J., Zeng,Z.C., Chen,R., Hyslop,T., Curran,W.J. Jr and Iliakis,G. (2000) DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. Cancer Res., 60, 1245–1253.[Abstract/Free Full Text]
  65. Chan,D.W. and Lees-Miller,S.P. (1996) The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit. J. Biol. Chem., 271, 8936–8941.[Abstract/Free Full Text]
  66. Critchlow,S.E., Bowater,R.P. and Jackson,S.P. (1997) Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr. Biol., 7, 588–598.[ISI][Medline]
  67. Leber,R., Wise,T.W, Mizuta,R. and Meek,K. (1998) The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase. J. Biol. Chem., 16, 1794–1801.
  68. Chan,D.W., Ye,R., Veillette,C.J. and Lees-Miller,S.P. (1999) DNA-dependent protein kinase phosphorylation sites in Ku 70/80 heterodimer. Biochemistry, 38, 1819–1828.[ISI][Medline]
  69. Janus,F., Albrechtsen,N., Wiesmuller,L., Grosse,F. and Deppert,W. (1999) The dual role model for p53 in maintaining genomic integrity. Cell. Mol. Life Sci., 55, 12–27.[ISI][Medline]
Received July 5, 2001; revised November 14, 2001; accepted January 11, 2002.