From the Department of Pharmacology, University of
Minnesota Medical School, Minneapolis, Minnesota 55455
Received for publication, September 20, 2000, and in revised form, November 22, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fanconi anemia (FA) is a genetic disorder
associated with genomic instability and cancer predisposition. Cultured
cells from FA patients display a high level of spontaneous chromosome
breaks and an increased frequency of intragenic deletions, suggesting that FA cells may have deficiencies in properly processing DNA double
strand breaks. In this study, an in vitro plasmid DNA end joining assay was used to characterize the end joining capabilities of
nuclear extracts from diploid FA fibroblasts from complementation groups A, C, and D. The Fanconi anemia extracts had 3-9-fold less DNA
end joining activity and rejoined substrates with significantly less
fidelity than normal extracts. Wild-type end joining activity could be
reconstituted by mixing FA-D extracts with FA-A or FA-C extracts, while
mixing FA-A and FA-C extracts had no effect on end joining activity.
Protein expression levels of the DNA-dependent protein
kinase (DNA-PK)/Ku-dependent nonhomologous DNA end-joining proteins Xrcc4, DNA ligase IV, Ku70, and Ku86 in FA and normal extracts
were indistinguishable, as were DNA-dependent protein kinase and DNA end binding activities. The end joining activity as
measured by the assay was not sensitive to the DNA-PK inhibitor wortmannin or dependent on the nonhomologous DNA end-joining factor Xrcc4. However, when DNA/protein ratios were lowered, the end joining
activity became wortmannin-sensitive and no difference in end joining
activity was observed between normal and FA extracts. Taken together,
these results suggest that the FA fibroblast extracts have a deficiency
in a DNA end joining process that is distinct from the
DNA-PK/Ku-dependent nonhomologous DNA end joining pathway.
Fanconi anemia (FA)1 is
an autosomal recessive disease characterized by developmental
abnormalities, progressive bone marrow failure, chromosomal
instability, and predisposition to cancer (1, 2). Somatic cell fusion
studies have demonstrated the existence of at least eight
complementation groups (FA-A through FA-H) (3). Presently, five of the
FA genes have been cloned, FANCA, FANCC,
FANCE, FANCF, and FANCG (4-8). The
biochemical functions of these proteins are unknown; thus, the
underlying defect of this disease has not been established.
In vitro analysis of cultured cells obtained from FA
patients reveals an elevated level of spontaneous chromosome breaks. The frequency of these chromosomal lesions is amplified following exposure to DNA cross-linking agents (1). FA cells also experience spontaneous and psoralen-induced DNA deletions at a higher frequency than normal cells. These DNA deletions have been detected both within
the endogenous hypoxanthine-guanine phosphoribosyltransferase gene and within a target gene present on an autonomously replicating plasmid (9, 10). These cellular phenotypes suggested that FA cells may
have deficiencies in processing DNA double strand breaks.
Recent reports have supported the hypothesis that FA cells have
deficiencies in rejoining double strand breaks (DSBs) (11, 12). In
these studies, linearized plasmid DNA was transfected into immortalized
FA lymphoblasts and recovered after 48 h. Analysis of
recircularized products revealed that the overall efficiency of plasmid
end joining was normal in FA lymphoblasts from complementation groups
B, C, and D, but error-free processing of blunt-ended substrates was
significantly compromised in these cells.
To gain further insight into the process of DNA end joining in FA
cells, we used an in vitro assay to examine the ability of
nuclear protein extracts prepared from diploid FA fibroblasts to rejoin
linear plasmid DNA substrates. Nuclear extracts from diploid
fibroblasts from patients from complementation groups A, C, and D had
3-9-fold less end joining activity and rejoined linear substrates
imprecisely at a higher frequency than extracts from normal donors.
This end joining deficiency was not due to the presence of an inhibitor
in the FA extracts or to deficiencies in proteins or activities known
to be involved in the well characterized DNA-PK/Ku nonhomologous DNA
end joining pathway (13). Wild-type end joining activity could be
reconstituted by mixing FA-D extracts with FA-A or FA-C extracts but
not by mixing FA-A with FA-C extracts. The end joining activity that
was deficient in the FA extracts was not sensitive to the DNA-PK
inhibitor wortmannin or dependent on Xrcc4. When a lower substrate
DNA/protein ratio was used in the end joining assay, the end joining
activity was wortmannin-sensitive, and indistinguishable end joining
levels were observed between normal and FA extracts.
Cell Culture and Conditions--
The diploid FA fibroblast cell
strains PD.134.F (FA-C), PD.220.F (FA-A), and PD.20.F (FA-D) as well as
the normal diploid cell strains PD.715.F, PD.13.F, PD.793.F, PD.792.F,
and PD.751.F were kindly provided by Dr. Markus Grompe (Oregon Health
Sciences University). These cells were maintained in minimum essential Nuclear Protein Extracts--
Nuclear extracts were prepared as
previously described (14). Briefly, cells harvested from confluent
100-mm tissue culture dishes were washed three times with ice-cold
phosphate-buffered saline and resuspended in 2 ml of hypotonic buffer A
(10 mM KCl, 10 mM Tris (pH 7.4), 10 mM MgCl2, and 10 mM dithiothreitol)
and kept on ice for 15 min. Phenylmethylsulfonyl fluoride was added to
1 mM, and cells were disrupted using a Dounce homogenizer
(20 strokes with a tight pestle). The released nuclei were pelleted and
resuspended in 2 ml of buffer A containing 350 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin,
1.0 µg/µl aprotinin, and 0.7 µg/ml pepstatin and incubated for
1 h on ice. The nuclei were centrifuged at 70,000 rpm in a Beckman
TL-100.3 rotor at 4 °C for 30 min, and the clear supernatant
was adjusted to 10% glycerol and 10 mM
End Joining Reactions--
DNA end joining reactions were
carried out essentially as described previously (16). Circular pCR 2.1 plasmid DNA (Invitrogen, Carlsbad, CA) was linearized by restriction
digestion with KpnI to create linear substrates with
3'-cohesive ends, EcoRI to produce 5'-cohesive ends, or
EcoRV to generate blunt-ended substrates (all endonucleases
were from New England Biolabs, Beverly, MA). After restriction digest,
substrates were ethanol-precipitated and resuspended in TE buffer, pH
8.0. 1 µg of linearized DNA was incubated with 5 µg of nuclear
protein extract in 70 mM Tris (pH 7.5), 10 mM
MgCl2, 10 mM dithiothreitol, and 1 mM ATP in a total volume of 50 µl. The reaction was
carried out at 14 °C for 12 h unless otherwise noted. The
reaction mixture was then treated with proteinase K at 37 °C for 30 min and electrophoretically separated on a 0.8% agarose gel in Tris
borate-EDTA buffer at 0.55 V/cm for 12-15 h. After staining in
ethidium bromide, gels were scanned on a Bio-Rad scanner using the
Molecular Analyst program and quantified using IP Lab Gel (Signal
Analytics Corp., Vienna, VA). A band that migrated with form II of the
uncut substrate DNA was labeled CC and called closed
circular; a band that migrated at the predicted size of a linear dimer
was labeled D; and bands that migrated larger than the
linear dimer were labeled as higher molecular weight products
(HM). To quantitate the percentage of rejoining, total
product formation was calculated (CC + D + HM) and divided by the sum of total substrate DNA in the
reaction (L + CC + D + HM).
Antibodies--
The anti-Ku70 and anti-Ku86 antibodies were
purchased from Serotech, (Raleigh, NC). The anti-Xrcc4 and anti-DNA
ligase IV antibodies were kindly provided by Drs. Susan Critchlow and
Stephen Jackson (Welcome/CRC Institute, Cambridge, UK). All primary
antibodies were raised in rabbits against recombinant human proteins.
Western Blot Analysis--
Protein samples (10 µg) were
resolved on SDS-polyacrylamide gels (17) and transferred to
nitrocellulose membranes (Bio-Rad). After a 1-h incubation in 5%
bovine serum albumin in Tris-buffered saline, the membrane was probed
with antibody (1:2000 dilution for anti-Ku70 and anti-Ku86 antibodies;
1:1000 dilution for anti-Xrcc4 and anti-DNA ligase IV antibodies). The
membrane was then washed three times in 0.1% bovine serum albumin in
Tris-buffered saline and incubated with diluted (1:5000) alkaline
phosphatase-conjugated goat-anti-rabbit IgG (Sigma) for 1 h at
room temperature. This was followed by three additional washes in 0.1%
bovine serum albumin. Incubation with Sigma Fast (Sigma), which
contains the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl
phosphate/nitro blue tetrazolium, was then carried out to detect the
presence of the proteins.
Electrophoretic Mobility Shift Assay--
A modification of the
assay used by Rathmell and Chu (18) was used to detect DNA end binding
activity in the nuclear extracts. A 394-base pair fragment from an
EcoRI digest of the plasmid pREP4 was radioactively
end-labeled with [ DNA-PK Assay--
The SignaTECT DNA-dependent
Protein Kinase Assay System (Promega, Madison, WI) was used to detect
DNA-PK activity following instructions provided by the manufacturer.
Wortmannin Inhibition--
A 0.1 mM stock of
wortmannin (Sigma) was prepared in 10% Me2SO.
Wortmannin was incubated with 5 µg of nuclear extracts for 30 min on
ice. Plasmid DNA end joining experiments as previously described were
then performed at 14 °C.
Immunodepletion--
Extracts (50 µg) were incubated with
antisera for 1 h at 4 °C on a rotary wheel. The
extract/antibody mixture was added to 25 µl of protein A-Sepharose
beads (Sigma) and incubated with rotation for 3 h at 4 °C. The
beads were removed by repeated centrifugation at 12,000 × g, and the supernatant was used for Western blot
analysis and end joining experiments.
Bacterial Transformation Assay--
End joining assays were
carried out as described above. After the 14 °C incubation, the
reaction mixture was incubated at 37 °C with calf intestinal
alkaline phosphatase (Roche Molecular Biochemicals) for 1 h.
Samples were then extracted with phenol/chloroform, ethanol-precipitated, and resuspended in 10 µl of TE buffer, pH 8.0. Electrocompetent E. coli (strain DH10B) was then
electroporated with 1 µl of recovered plasmid DNA using a Life
Technologies, Inc. Gene Pulser at a field strength of 2.44 kV/cm and
plated on ampicillin-containing LB plates containing
isopropyl-1-thio- To test the end joining activity of FA fibroblasts, an in
vitro DNA end joining assay that has been previously described was employed (16). Nuclear protein extracts were prepared from diploid fibroblasts from normal donors and diploid fibroblasts from FA patients
of complementation groups A (FA-A), C (FA-C), and D (FA-D). 5 µg of
extract was incubated for 12 h at 14 °C with 1 µg of plasmid pCR2.1 DNA that had been linearized by restriction digestion to have
either blunt or cohesive ends. End joining activity was detected by the
presence of closed circular, linear substrate, and high molecular
weight products when the reaction mixture was analyzed by agarose gel
electrophoresis. In end joining experiments performed with blunt-ended
substrate, closed circular was the predominant product formed. When
cohesive-ended substrates were used, closed circular, linear substrate,
and high molecular weight products were detected on the ethidium
bromide-stained gels.
Fig. 1a shows the results of
an end joining experiment performed with blunt-ended substrate.
Scanning laser densitometry was used to quantitate the bands, and the
percentage of linear substrate that had been rejoined was determined.
This analysis revealed that the normal extract rejoined 34% of the
linearized substrate as compared with 3, 7, and 7%, respectively, by
the FA-A, FA-C, and FA-D extracts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-medium supplemented with 2 mM glutamine and 15% fetal
bovine serum. Normal diploid strains CRL-2115, CRL-2068, and CRL-2072 were purchased from the American Type Culture Collection (Manassas, VA)
and were maintained in Eagle's minimum essential medium
supplemented with 2 mM glutamine, 1 mM sodium
pyruvate, and 10% fetal bovine serum. All cells were maintained at
37 °C in a humidified, 5% CO2 environment.
-mercaptoethanol. The resulting extracts were dialyzed against a
buffer containing 25 mM Tris (pH 7.5), 1 mM
EDTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and 10% glycerol. Protein
concentrations were determined by the Bradford method (15).
-32P]dATP by using the Klenow
fragment of Escherichia coli DNA polymerase. 0.2 ng of probe
was incubated with 0.3 µg of nuclear extract in 12 mM
HEPES, 5 mM MgCl2, 4 mM Tris (pH
7.9), 100 mM KCl, 0.6 nM EDTA, 0.6 mM dithiothreitol, and 6% glycerol in the presence of 200 ng of unlabeled supercoiled DNA. To specifically compete for the DNA
end binding activity, 200 ng of unlabeled linear plasmid DNA was added
in place of circular DNA to the reaction mix. Reactions were carried
out at 14 °C for 30 min in a final volume of 10 µl. Following
incubation, 5× loading dye (0.25% bromphenol blue, 0.25% xylene
cyanol, 30% glycerol) was added to each reaction and subjected to
electrophoresis on a 5% polyacrylamide gel in TBE (90 mM
Tris borate, 2 mM EDTA) running buffer. The gel was run at
20 V/cm for 2 h at 4 °C. Detection of radioactivity was
achieved using a PhosphorImager (Molecular Dynamics, Inc., Foster City, CA).
-D-galactopyranoside and
5-bromo-4-chloro-3-indoyl-
-D-galactoside. Bacterial
transformants containing imprecisely end-joined plasmids were detected
as white colonies, whereas transformants harboring precisely rejoined
plasmids formed blue colonies. Plasmid DNA was isolated by the alkaline lysis method, characterized by restriction mapping, and sequenced using
a PerkinElmer Life Sciences automated DNA sequencer (Microchemical Facility, University of Minnesota).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
[in a new window]
Fig. 1.
End joining activity of DNA substrates with
blunt and cohesive ends is decreased in nuclear extracts prepared from
FA-A, FA-C, and FA-D fibroblasts. A, end joining of DNA
substrates with blunt ends was performed as described under
"Experimental Procedures." Samples were loaded on a 0.8% agarose
gel, electrophoretically resolved, and stained with ethidium bromide.
, no extract; N, 5 µg of nuclear protein extract from
the normal cell strain PD.715.F; A, 5 µg of nuclear
extract from FA-A cell strain PD.220.F; C, 5 µg of nuclear
extract from FA-C cell strain PD.134.F; D, 5 µg of nuclear
extract from FA-D cell strain PD.20.F. L represents the
mobility of the linear substrate DNA, and CC indicates the
mobility of a product that migrates with form II of uncut substrate DNA
and we call "closed circular." B, DNA end joining
reactions were performed with blunt and cohesive-ended substrates with
extracts from eight normal diploid fibroblast strains (N),
FA-A cell strain PD.220.F (A), FA-C cell strain PD.134.F
(C), and FA-D cell strain PD.20.F (D). Scanning
laser densitometry was performed to determine the percentage of plasmid
substrate that had been converted to product. The mean end joining
activity for the normal fibroblast extracts was calculated from the
mean end joining activities in eight normal fibroblast strains. The
mean end joining activities in the extracts from the FA-A, FA-C, and
FA-D cell strains were determined from multiple end joining experiments
(FA-A, n = 4; FA-C, n = 5; FA-D,
n = 3) performed with a minimum of two independently
prepared extracts. The bars represent the mean percentage
end joining ± S.E. Filled bars, blunt-ended
substrates; open bars, 5'-cohesive-ended
substrate; *, p < 0.001; #, p < 0.05.
To confirm that the FA-A, FA-C, and FA-D extracts were deficient in DNA end joining, two or three independent extracts were prepared from each cell line and tested multiple times in end joining experiments with blunt and cohesive-ended substrates. Also, nuclear extracts were prepared from seven normal fibroblast strains in addition to the normal fibroblast strain used in Fig. 1a (PD.715.F) and tested for end joining activity. Fig. 1b depicts the average end joining activity (percentage rejoined ± S.E.) in the normal, FA-A, FA-C, and FA-D extracts. The mean end joining activity of blunt-ended substrate in extracts from the normal strains ranged from 19 to 45% with a cumulative mean of 31 ± 4%. Rejoining of cohesive-ended substrate by the normal extracts ranged from 16 to 37% (cumulative mean, 26 ± 3%). The normal strain, PD.715.F, used in Fig. 1a and subsequent figures, rejoined 41 ± 3% of blunt-ended substrate and 35 ± 4% of cohesive-ended substrate. In comparison, the extracts from the FA-A cell strain rejoined 4 ± 1% of both substrates, the extracts from the FA-C cell strain rejoined 6 ± 2% of blunt-ended substrate and 12 ± 2% of cohesive-ended substrate, and the extracts from the FA-D cells rejoined 15 ± 3% and 10 ± 2% of the blunt- and cohesive-ended substrates, respectively. Comparison of the mean end joining activities in the eight normal fibroblast strains with the mean end joining activities in the FA extracts revealed that the FA-A, FA-C, and FA-D extracts were significantly less able to rejoin linearized plasmid substrates with blunt and cohesive ends than the normal extracts.
In the above experiments, the cohesive-ended substrate had a 5'-overhang. To determine whether the FA extracts were also deficient at rejoining substrates with 3'-cohesive ends, end joining experiments were performed with a substrate that was linearized by restriction digest with KpnI. Nuclear extracts from the eight normal fibroblast strains rejoined a mean of 48 ± 4% of the substrate, with a range from 35 to 64%. The FA-A, FA-C, and FA-D extracts rejoined 3, 2, and 12%, respectively, of the 3'-cohesive-ended substrate (data not shown). We concluded that FA-A, FA-C, and FA-D extracts were deficient in rejoining plasmid DNA substrates with blunt-, 5'-cohesive, and 3'-cohesive ends.
To determine whether the observed end joining deficiency in the FA
extracts was due to reduced kinetics of activity, a time course
experiment was performed with normal and FA-C extracts. The plasmid end
joining assay was performed using a blunt-ended substrate, and aliquots
were removed for analysis at 4, 8, and 12 h. As shown in Fig.
2a, the normal cell extract
rejoined the blunt-ended substrate in a linear fashion between 4 h
(25% rejoined) and 8 h (44% rejoined) before reaching saturation
at 12 h (52% rejoined). On the other hand, no activity was
detected in samples incubated with the FA fibroblast extract for up to
8 h. Following a 12-h incubation period, 4% rejoining was
detected. Similarly, we tested the end joining activity of the normal
and the FA-C fibroblast extracts as a function of protein
concentration. Plasmid DNA end joining with blunt-ended substrate was
performed using 2.5, 5, 7.5, 10, and 12.5 µg of protein. As seen in
Fig. 2b, there was a noticeable difference in end joining
activity between the two extracts at all protein concentrations tested.
Similar observations were made when cohesive-ended substrates or FA-A
and FA-D extracts were used (data not shown).
|
Deficient end joining capabilities of the FA extracts can be explained
by two alternate hypotheses; either the extracts derived from the FA
fibroblasts contain an inhibitor of the end joining reaction, or,
conversely, a factor or factors essential for the end joining reaction
may be absent from the FA extracts. To distinguish between these two
possibilities, an end joining experiment with blunt-ended substrate was
performed using a mixture of equal amounts of nuclear protein extract
from normal and FA-C cells. To keep the total protein present in the
reaction at 5 µg, 2.5 µg of the normal extract was mixed with 2.5 µg of the FA extract. As seen in Fig.
3, 5 µg of normal extract alone yielded
42% product formation. When a mixture of normal and FA extracts was
tested, 34% product formation was detected. This level of end joining
is consistent with the percentage of end joining obtained when 2.5 µg
of normal extract is used in end joining reactions (see Fig.
2b). The same results were obtained when FA-A or FA-D
extracts were mixed with normal extracts or when cohesive-ended
substrates were used (data not shown). The wild-type level of plasmid
end joining activity present in the mixed sample is inconsistent with
the notion that an inhibitor of end joining is present in the FA
nuclear extract. This finding indicates that nuclear extracts from FA
fibroblasts lack a factor or factors essential for the end joining
reaction.
|
The reduced DNA end joining activity in extracts prepared from FA-A,
FA-C, and FA-D fibroblast strains raised the possibility that wild-type
end joining activity could be reconstituted by mixing combinations of
the FA extracts. We therefore mixed 2.5 µg of extract from FA-A,
FA-C, and FA-D fibroblasts in combination with one another and
performed DNA end joining experiments. As seen in Fig.
4, 5 µg of normal extract alone
rejoined 33% of the linearized DNA substrate. Combining 2.5 µg of
FA-A or FA-C extracts with 2.5 µg of FA-D extract resulted in 29 and
32%, respectively, of DNA end joining activity, whereas a mixture of
the FA-A and FA-C extracts had no effect on end joining levels (7% end
joining activity).
|
It has been previously established that immortalized FA lymphoblasts
rejoin blunt-ended DNA substrates in an error-prone manner in
vivo relative to control lymphoblasts (12, 13). We therefore examined the nature of DNA end joining in nuclear extracts from FA-C
fibroblasts and nuclear extracts from normal donors. A bacterial transformation assay was employed to analyze the fidelity of end joining in the extracts (see "Experimental Procedures"). Using blunt-ended substrate, a significantly higher number of white colonies
were recovered from rejoining by the FA-C extract as compared with the
normal extract, 27% (217 of 588) versus 18% (171 of 780)
(p < 0.005, 2 = 36.67) (Fig.
5). Similarly, a significantly higher
number of white colonies were recovered from the FA-C extract when
cohesive-ended substrates were used. 4% (33 of 899) of the colonies
resulting from the rejoining by the FA-C extract were white compared
with 0.5% (15 of 2917) from the normal fibroblast extract. Statistical analysis again revealed that the difference was significant
(p < 0.005,
2 = 52.8).
|
Restriction digest analysis of imprecisely rejoined plasmids from the FA-C (101 plasmids) and normal (69 plasmids) end joining experiments revealed that all imprecise rejoining events resulted in deletions (data not shown). Sequence data of the region flanking the rejoining site obtained from 26 plasmids that resulted from imprecise rejoining (13 from FA and 13 from normal extracts) showed no difference in the range of the sizes of the deletions (2-47 base pairs). It also showed that all deletions, irrespective of whether obtained from the FA-C or normal extract, were flanked by direct repeat sequences of between 1 and 7 base pairs in length (data not shown). From this analysis, we concluded that there was no difference in the nature of the imprecise end joining events between the normal and FA-C extracts.
Currently, two independent pathways are known to repair DSBs in cells: homologous recombination and nonhomologous DNA end joining (NHEJ). In contrast to genetic screens of x-ray-sensitive yeast cells, which identified genes involved in homologous recombination (HR) (19, 20), mutant screens of x-ray-hypersensitive mammalian cells have led to the identification of factors involved in NHEJ (21). This led to the suggestion that DSBs are preferentially repaired by NHEJ in mammalian cells (13).
A NHEJ pathway, generally referred to as the DNA-PK- or
Ku-dependent pathway, has been well characterized in recent
years. It has been demonstrated to be minimally dependent upon five
proteins: Xrcc4, DNA ligase IV, Ku70, Ku86, and the catalytic subunit
of DNA-dependent protein kinase (DNA-PKcs)
(22). To test if any of these proteins were absent from FA-C extracts,
Western blot analysis was performed using antisera specific for the
Ku70, Ku86, DNA ligase IV, and Xrcc4 proteins. The levels of these four
proteins present in nuclear extracts from FA-C fibroblasts were
essentially identical to those seen in the normal fibroblast extract
(Fig. 6a), suggesting that low
expression of any of these four proteins was not the cause of the
deficient end joining in the FA extracts.
|
The presence of wild-type levels of these proteins does not prove that they are functional. However, the amount of DNA end binding activity, which is dependent upon the functional Ku70/Ku86 heterodimer as determined by the ability of Ku70 and Ku86 antibodies to supershift the end binding band (23), was identical in control and FA-C extracts (Fig. 6b). Similarly, DNA-PK activities in FA-C extracts were indistinguishable from those present in extracts from control fibroblasts (not shown). Western blot analysis, DNA end binding, and DNA-PK assays performed with FA-A and FA-D extracts revealed no differences from normal fibroblasts in these extracts as well (data not shown). Previous studies performed with FA lymphoblasts also found no deficiencies in these factors or activities (11, 12).
We next wished to determine whether the assay used in this study was
measuring NHEJ activity. To do this, we determined if the end joining
activity was dependent on DNA-PK or Xrcc4, two components of the
DNA-PK/Ku-dependent NHEJ pathway (22). Wortmannin is a
potent inhibitor of phosphatidylinositol 3-kinases (24) and has been
demonstrated to inhibit DNA-PK activity (25). 5.0 µg of nuclear
extract prepared from normal diploid fibroblasts was incubated for 30 min on ice with 5.0 µM wortmannin. This concentration of
wortmannin abolished DNA-PK activity in the extract as measured by an
in vitro DNA-PK activity kit (Fig.
7a). DNA end joining reactions
with 5'-cohesive-ended substrates were then performed at 14 °C for
4 h. Wortmannin had no inhibitory effects on the end joining
activity of these extracts (Fig. 7b). Wortmannin also had no
inhibitory effects on the end joining activity detected in FA extracts
(Fig. 7c).
|
To determine whether Xrcc4 was required for the end joining activity,
antiserum raised against the human Xrcc4 protein was used to
immunodeplete Xrcc4 from a nuclear extract prepared from fibroblasts
from a normal donor. Western blot analysis confirmed that the extract
was depleted of this protein (Fig.
8A). DNA end joining
experiments were performed at 14 °C for 4 h with the
Xrcc4-immunodepleted extract. As shown in Fig. 8B, the
Xrcc4-immunodepleted extract had wild-type end joining activity. We
concluded that the end joining assay as used in this study was
measuring an activity that was not dependent on the NHEJ factors DNA-PK
or Xrcc4.
|
Baumann and West described an in vitro end joining assay
that was dependent on the NHEJ factors Ku70, Ku86, Xrcc4, DNA ligase IV, and DNA-PKcs (22). Under the conditions of this assay,
10-100 ng of substrate DNA was incubated with 20-60 µg of cellular
extract (0.001 µg of DNA/µg of protein as compared with 0.2 µg of
DNA/µg of protein in the current study). To more closely resemble the conditions described by Baumann and West, we performed end joining experiments for 2 h at 14 °C using 50 ng of linearized DNA and 5 µg of nuclear extracts (0.05 µg of DNA/µg of protein) from
normal and FA fibroblasts. As shown in Fig.
9, similar to what was reported by
Baumann and West, the end joining activity was sensitive to wortmannin
under these conditions. The end joining activity observed in the normal
and FA extract were identical under these conditions. The substrate DNA
concentration was the only variable changed under the
wortmannin-sensitive conditions, suggesting that the DNA/protein ratio
is crucial in achieving a DNA-PK-dependent end joining
activity.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The data presented here demonstrate that extracts from diploid Fanconi anemia fibroblasts are deficient in end joining blunt and cohesive-ended linear plasmid DNA substrates. The end joining deficiency appears to be a common feature of FA, since extracts from fibroblasts from complementation groups A, C, and D share the defect.
Several lines of in vitro evidence indicate that eukaryotic cells rely upon more than one DNA end joining pathway. In a study performed by Johnson and Fairman (26), calf thymus extracts were fractionated into four biochemically distinct fractions. Despite the presence of Ku70 in only one of the fractions, end joining activity was detected in all fractions. In a second study, extracts that were prepared from the DNA-PK mutant tumor cell line MO59 had wild-type end joining activity, suggesting that a DNA-PK-independent end joining pathway was functioning in these cells (27).
In vivo studies have also supported the hypothesis that multiple DNA end joining pathways exist. Genetic inactivation studies carried out in the yeast Saccharomyces cerevisiae convincingly demonstrate that this organism repairs DNA double strand breaks through precise (Ku-dependent) and imprecise (Ku-independent) pathways (28, 29). The same appears to be true in mammalian cells. Both wild-type and xrs6 hamster cells, which lack a functional Ku86 protein, were able to rejoin transfected linear plasmids. However, rejoining occurred in a more error prone manner in the xrs6 cells, and the deletions formed were on average 3-4-fold larger than in control cells (30). Taken together, these in vitro and in vivo studies strongly suggest that more than one NHEJ pathways exist.
The data presented in this study suggest that the FA extracts are deficient in a DNA end joining pathway that is distinct from the well characterized NHEJ pathway. First, FA extracts have wild type levels of the NHEJ factors Xrcc4, DNA ligase IV, Ku70, and Ku86 as well as normal levels of DNA-PK and DNA end binding activities. Second, the end joining activity in which the FA extracts are deficient is not inhibited by the DNA-PK inhibitor wortmannin. Third, extracts immunodepleted of Xrcc4 have wild-type end joining activity as measured by the assay. Finally, when end joining reactions are carried out similar to those previously reported for measuring NHEJ (22), there is no difference in end joining between normal and FA extracts, indicating that the well characterized NHEJ pathway is fully functional in the FA extracts.
The possibility cannot be ruled out that Ku is involved in this alternate end joining process. However, given that FA extracts have wild-type DNA end binding activity, it seems unlikely that the defect in FA cells is the result of defective Ku proteins. In addition, cells with Ku defects are sensitive to ionizing radiation (13, 21), a phenotype not associated with FA cells (31). Regardless of the involvement of Ku in this pathway, the data indicates that the end joining activity that is deficient in the FA extracts is independent of DNA-PK and Xrcc4. Thus, this end joining pathway appears to be distinct from that classically referred to as NHEJ.
It is tempting to speculate that the DNA end joining deficiency observed in FA extracts is representative of a cellular defect in rejoining double strand breaks. This alternate end joining pathway may represent an additional end joining pathway in cells. This could explain why end joining activity is still detected in Ku-deficient cells (28-30). This could also explain why Escarceller et al. (11, 12) found no deficiency in the overall end joining efficiency of linearized plasmid substrates but did see error-prone rejoining of blunt-ended substrates in FA lymphoblasts. In vivo, a defect in this additional end joining pathway would be masked by other DNA end joining pathways.
An end joining deficiency in FA cells could account for many of the cellular phenotypes associated with this disorder such as high levels of spontaneous and DNA cross-link-induced chromosomal breaks and high frequencies of spontaneous and psoralen-induced deletions. An end joining deficiency could also explain the predisposition to cancer associated with these patients, since unrepaired or misrepaired DNA lesions could ultimately lead to loss of function of genes essential for proper cellular maintenance and growth.
In addition, a DNA end joining defect in FA cells could potentially explain a previous result observed by our laboratory. HR activity was found to be elevated in Fanconi anemia fibroblasts and in nuclear extracts prepared from FA cells as compared with HR activity in fibroblasts from normal donors (32). We have also observed that Rad51, the mammalian homologue of the bacterial recombination protein RecA, is substantially elevated in extracts prepared from FA fibroblasts.2 Recently, it was demonstrated that the human Rad52 protein, a protein involved in mammalian HR, binds double strand breaks (33). We speculate that HR and Rad51 may be elevated in FA cells in response to an increased number of unrepaired DSBs that result from the described end joining deficiency.
Finally, the data indicate that mixing FA-A or FA-C extracts with FA-D
extracts is able to reconstitute wild-type end joining activity levels,
while mixing FA-A and FA-C extracts has no effect on end joining
levels. While FA-D patients have the same clinical symptoms as FA
patients from the other complementation groups, there are reports of
FA-D cells having unique biochemical characteristics (34-36). In
particular, a multiprotein complex of four cloned FA gene products
(FANCA, FANCC, FANCG, and FANCF) is detected only in wild-type and FA-D
cells, indicating that all of the FA proteins, with the exception of
FANCD, are required for the proper formation of this complex (36). One
could imagine that a preassembled "FA complex" is required for
wild-type end joining activity in the extracts. This could only be
provided by extracts from FA-D cells. Thus, when FA-A and FA-C extracts
are mixed, neither would provide the "FA complex," and wild-type
end joining activity would not be reconstituted.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the National Institutes of Health (CA61906 and AG16678) and the American Heart Association (9951198Z).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: CRI, Center III, Children's National Medical Center, 111 Michigan Ave. NW, Washington, D. C. 20010.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church St., SE, Minneapolis, MN 55455. E-mail: campb034@maroon.tc.umn.edu.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M008634200
2 R. Lundberg and C. Campbell, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: FA, Fanconi anemia; DSB, double strand break; NHEJ, nonhomologous DNA end joining; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, catalytic subunit of DNA-PK; HR, homologous recombination.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Auerbach, A. D., and Allen, R. G. (1991) Cancer Genet. Cytogenet. 51, 1-12[CrossRef][Medline] [Order article via Infotrieve] |
2. |
D'Andrea, A. D.,
and Grompe, M.
(1997)
Blood
90,
1725-1736 |
3. | Joenje, H., Oostra, A., Wijker, M., di Summa, F., van Berkel, C., Rooimans, M., Ebell, W., van Weel, M., Pronk, J., Buchwald, M., and Arwert, F. (1997) Am. J. Hum. Genet. 61, 940-944[Medline] [Order article via Infotrieve] |
4. | Lo Tenfoe, J. R., Rooimans, M. A., Bosnoyan-Collins, L., Alon, N., Wijker, M., Parker, L., Lightfoot, J., Carreau, M., Callen, D. F., Savoia, A., Cheng, N. C., Vanberkel, C. G. M., Strunk, H. M. P., Gille, J. J. P., Pals, G., Kruyt, F. A. E., Pronk, J. C., Arwert, F., Buchwald, M., and Joenje, H. (1996) Nat. Genet. 14, 320-323[Medline] [Order article via Infotrieve] |
5. | Strathdee, C. A., Gavish, H., Shannon, W. R., and Buchwald, M. (1992) Nature 356, 763-767[CrossRef][Medline] [Order article via Infotrieve] |
6. | de Winter, J. P., Leveille, F., van Berkel, C. G. M., Rooimans, M. A., van der Weel, L., Steltenpool, J., Deuth, I., Morgan, N. V., Alon, N., Bosnoyan-Collins, L., Lightfoot, J., Leegwater, P. A., Waisfisz, Q., Komatsu, K., Arwert, F., Pronk, J. C., Mathew, C. G., Digweed, M., Buchwald, M., and Joenje, H. (2000) Am. J. Hum. Genet. 67, 1305-1308 |
7. | de Winter, J. P., Rooimans, M. A., van der Weel, L., van Berkel, C. G. M., Alon, N., Bosnoyan-Collins, L., de Groot, J., Zhi, Y., Waisfisz, Q., Pronk, J. C., Arwert, F., Mathew, C. G., Scheper, R. J., Hoatlin, M. E., Buchwald, M., and Joenje, H. (2000) Nat. Genet. 24, 15-16[CrossRef][Medline] [Order article via Infotrieve] |
8. | de Winter, J. P., Waisfisz, Q., Rooimans, M. A., van Berkel, C. G. M., Bosnoyan-Collins, L., Alon, N., Carreau, M., Bender, O., Demuth, I., Schindler, D., Pronk, J. C., Arwert, F., Hoehn, H., Digweed, M., Buchwald, M., and Joenje, H. (1998) Nat. Genet. 20, 281-283[CrossRef][Medline] [Order article via Infotrieve] |
9. | Bredberg, A., Sandor, Z., and Brant, M. (1995) Carcinogenesis 16, 555-561[Abstract] |
10. | Papadopoulo, D., Guillouf, C., Mohrenweiser, H., and Moustacchi, E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8383-8387[Abstract] |
11. | Escarceller, M., Rousset, S., Moustacchi, E., and Papadopoulo, D. (1997) Somatic Cell Mol. Genet. 23, 401-411[Medline] [Order article via Infotrieve] |
12. | Escarceller, M., Buchwald, M., Singleton, B. K., Jeggo, P. A., Jackson, S. P., Moustacchi, E., and Papadopoulo, D. (1998) J. Mol. Biol. 279, 375-385[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Chu, G.
(1997)
J. Biol. Chem.
272,
24097-24100 |
14. | Jessberger, R., and Berg, P. (1991) Mol. Cell. Biol. 11, 445-457[Medline] [Order article via Infotrieve] |
15. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
16. | North, P., Ganesh, A., and Thacker, J. (1990) Nucleic Acids Res. 18, 6205-6210[Abstract] |
17. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
18. | Rathmell, W. K., and Chu, G. (1994) Mol. Cell. Biol. 14, 4741-4748[Abstract] |
19. | Game, J. C. (1993) Semin. Cancer Biol. 4, 73-83[Medline] [Order article via Infotrieve] |
20. | Thompson, L. H., and Schild, D. (1999) Biochimie (Paris) 81, 87-105[CrossRef][Medline] [Order article via Infotrieve] |
21. | Zdzienicka, M. Z. (1995) Mut. Res. 336, 203-213[Medline] [Order article via Infotrieve] |
22. |
Baumann, P.,
and West, S. C.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14066-14070 |
23. |
Coffey, C.,
Lakshmipathy, U.,
and Campbell, C.
(1999)
Nucleic Acids Res.
27,
3348-3354 |
24. | Powis, G., Bonjouklian, R., Berggren, M. M., Gallegos, A., Abraham, R., Ashendel, C., Zalkow, L., Matter, W. F., Dodge, J., Grindley, G., and Vlahos, C. J. (1994) Cancer Res. 54, 2419-2423[Abstract] |
25. | Hartley, K. O., Gell, D., Smith, G. C., Zhang, H., Divecha, N., Connelly, M. A., Admon, A., Lees-Miller, S. P., Anderson, C. W., and Jackson, S. P. (1995) Cell 82, 849-856[Medline] [Order article via Infotrieve] |
26. | Johnson, A. P., and Fairman, M. P. (1996) Mut. Res. 364, 103-116[Medline] [Order article via Infotrieve] |
27. | Cheong, N., Perrault, A. R., Wang, H., Wachsberger, P., Mammen, P., Jackson, I., and Iliakis, G. (1999) Int. J. Radiat. Biol. 75, 67-81[CrossRef][Medline] [Order article via Infotrieve] |
28. | Boulton, S. J., and Jackson, S. P. (1996) EMBO J. 15, 5093-5103[Abstract] |
29. | Wilson, T. E., Grawunder, U., and Lieber, M. R. (1997) Nature 388, 495-498[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Liang, F.,
and Jasin, M.
(1996)
J. Biol. Chem.
271,
14405-14411 |
31. | Duckworth-Rysiecki, G., and Taylor, A. M. (1985) Cancer Res. 45, 416-420[Abstract] |
32. |
Thyagarajan, B.,
and Campbell, C.
(1997)
J. Biol. Chem.
272,
23328-23333 |
33. | Van Dyck, E., Stasiak, A. Z., Stasiak, A., and West, S. C. (1999) Nature 398, 728-731[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Yamashita, T.,
Kupfer, G. M.,
Naf, D.,
Suliman, A.,
Joenje, H.,
Asano, S.,
and D'Andrea, A. D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13085-13090 |
35. |
Garcia-Higuera, I.,
Kuang, Y.,
Naf, D.,
Wasik, J.,
and D'Andrea, A.
(1999)
Mol. Cell. Biol.
19,
4866-4873 |
36. |
de Winter, J. P.,
van der Wall, L.,
de Groot, J.,
Stone, S.,
Waisfisz, Q.,
Arwert, F.,
Scheper, R. J.,
Kruyt, F. A. E.,
Hoatlin, M. E.,
and Joenje, H.
(2000)
Hum. Mol. Genet.
9,
2665-2674 |