Deficient DNA end-joining activity in extracts from Fanconi anemia fibroblasts Richard Lundberg1 ...

46 Pages·2000·0.14 MB·English

by  Tim Walseth

JBC Papers in Press. Published on December 21, 2000 as Manuscript M008634200
Deficient DNA end-joining activity
in extracts from Fanconi anemia fibroblasts
Richard Lundberg1, Manohara Mavinakere1, 2, and Colin Campbell1
1Department of Pharmacology, University of Minnesota Medical School
6-120 Jackson Hall, 321 Church Street, SE, Minneapolis, MN 55455
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2Current address: CRI, Center III, Children’s National Medical Center, 111 Michigan nlo
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Ave. NW, Washington, D. C. 20010 fro
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Correspondence should be addressed to C. C. email: [email protected]
Running Title: DNA end-joining in Fanconi anemia fibroblast extracts
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
DNA end-joining in Fanconi anemia fibroblast extracts
Summary
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
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normal extracts. Wild-type end-joining activity could be reconstituted by mixing FA-D o
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extracts with FA-A or FA-C extracts, while mixing FA-A and FA-C extracts had no e
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effect on end-joining activity. Protein expression levels of the DNA-dependent protein h
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kinase (DNA-PK)/Ku-dependent non-homologous DNA end-joining proteins Xrcc4, w
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DNA ligase IV, Ku70, and Ku86 in FA and normal extracts were indistinguishable, as .org
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were DNA-dependent protein kinase and DNA end-binding activities. The end-joining u
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activity as measured by the assay was not sensitive to the DNA-PK inhibitor wortmannin A
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or dependent on the NHEJ factor Xrcc4. However, when DNA/protein ratios were , 2
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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 NHEJ pathway.
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DNA end-joining in Fanconi anemia fibroblast extracts
Introduction
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.
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In vitro analysis of cultured cells obtained from FA patients reveals an elevated o
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level of spontaneous chromosome breaks. The frequency of these chromosomal lesions e
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is amplified following exposure to DNA cross-linking agents (1). FA cells also h
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experience spontaneous and psoralen-induced DNA deletions at a higher frequency than w
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normal cells. These DNA deletions have been detected both within the endogenous .org
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HPRT gene and within a target gene present on an autonomously replicating plasmid (9, u
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10). These cellular phenotypes suggested that FA cells might have deficiencies in A
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processing DNA double-strand breaks. , 2
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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 hours.
Analysis of re-circularized 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.
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DNA end-joining in Fanconi anemia fibroblast extracts
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 re-join 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, nor to deficiencies in proteins or activities known to be
involved in the well-characterized DNA-PK/Ku non-homologous DNA end-joining
pathway (13). Wild-type end-joining activity could be reconstituted by mixing FA-D
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extracts with FA-A or FA-C extracts, but not by mixing FA-A with FA-C extracts. The lo
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end-joining activity that was deficient in the FA extracts was not sensitive to the DNA- fro
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PK inhibitor wortmannin or dependent on Xrcc4. When a lower substrate DNA/protein ://w
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ratio was used in the end-joining assay, the end-joining activity was wortmannin- .jb
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sensitive and indistinguishable end-joining levels were observed between normal and FA b/
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extracts. t o
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DNA end-joining in Fanconi anemia fibroblast extracts
Experimental Procedures
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 Alpha Medium supplemented with 2mM 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
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maintained in Minimum Essential Medium-Eagle supplemented with 2 mM glutamine, 1 lo
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mM sodium pyruvate, and 10% fetal bovine serum. All cells were maintained at 37 (cid:176)C in fro
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a humidified, 5% CO2 environment. ://w
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Nuclear protein extracts- Nuclear extracts were prepared as previously described (14). .jb
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Briefly, cells harvested from confluent 100 mm tissue culture dishes were washed 3 times b/
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with ice-cold phosphate buffered saline and resuspended in 2 ml of hypotonic buffer A t o
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(10 mM KCL, 10 mM Tris [pH 7.4], 10 mM MgCl2, and 10 mM dithiothreitol) and kept ril 11
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on ice for 15 minutes. Phenylmethylsulfonyl fluoride (PMSF) was added to 1mM and 19
cells were disrupted using a Dounce homogenizer (20 strokes with a tight pestle). The
released nuclei were pelleted and resuspended in 2 ml buffer A containing 350 mM NaCl,
1mM PMSF, 0.5 µg/ml leupeptin, 1.0 µg/ul aprotinin, and 0.7 µg/ml pepstatin and
incubated for 1 hour on ice. The nuclei were centrifuged at 70,000 rpm in a Beckman
TL-100.3 rotor at 4 (cid:176) C for 30 minutes and the clear supernant was adjusted to 10%
glycerol and 10mM -b mercaptoethanol. The resulting extracts were dialyzed against a
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DNA end-joining in Fanconi anemia fibroblast extracts
buffer containing 25 mM Tris [pH 7.5], 1 mM EDTA, 1 mM DTT, 1mM PMSF, and
10% glycerol. Protein concentrations were determined by the Bradford method (15).
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 Kpn I to create linear substrates with 3’-
cohesive ends, EcoR I to produce 5’-cohesive ends, or EcoR V 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 mg of linearized DNA was incubated with 5 m g of nuclear protein extract in 70
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mM Tris [pH 7.5], 10 mM MgCl , 10 mM DTT, and 1mM ATP in a total volume of 50 lo
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ml. The reaction was carried out at 14 (cid:176) C for 12 hours unless otherwise noted. The fro
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reaction mixture was then treated with proteinase K at 37 (cid:176) C for 30 minutes and ttp://w
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electrophoretically separated on a 0.8% agarose gel in Tris-borate-EDTA buffer at 0.55 .jb
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V/cm for 12-15 hours. After staining in ethidium bromide, gels were scanned on a Bio- by/
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Rad scanner using the Molecular Analyst program and quantified using IP Lab Gel t o
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(Signal Analytics Corporation, Vienna, WV). A band that migrated with form II of the ril 1
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uncut substrate DNA was labeled CC and called closed circular, a band that migrated at 9
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
percent 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
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DNA end-joining in Fanconi anemia fibroblast extracts
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 mg) were resolved on SDS-polyacrylamide
gels (17) and transferred to nitrocellulose membranes (Bio-Rad). After a one-hour
incubation in 5% BSA in Tris-buffered saline (TBS) the membrane was probed with
antibody (1:2000 dilution for anti-Ku70 and anti-Ku80 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 (BSA) in TBS and incubated with diluted (1:5000)
alkaline phosphatase conjugated goat-anti-rabbit IgG (Sigma, St. Louis, MO) for one
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hour at room temperature. This was followed by 3 additional washes in 0.1% BSA. lo
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Incubation with Sigma Fast (Sigma, St. Louis, MO), which contains the alkaline fro
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phosphatase substrate BCIP/NBT, was then carried out to detect the presence of the ://w
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proteins. .jb
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Electrophoretic mobility shift assay- A modification of the assay used by Rathmell and b/
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Chu (18) was used to detect DNA end-binding activity in the nuclear extracts. A 394 t o
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base pair fragment from an EcoR I digest of the plasmid pREP4 was radioactively end- ril 1
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labeled with [a -32P] dATP by using the Klenow fragment of E. coli DNA polymerase. 19
0.2 ng of probe was incubated with 0.3 m g nuclear extract in 12 mM HEPES, 5 mM
MgCl , 4 mM Tris [pH 7.9], 100 mM KCl, 0.6 nM EDTA, 0.6 mM DTT, and 6 %
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glycerol in the presence of 200 ng 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 (cid:176) C for 30
minutes in a final volume of 10ml. Following incubation, 5x loading dye (0.25%
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DNA end-joining in Fanconi anemia fibroblast extracts
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 hours at 4 (cid:176) C. Detection
of radioactivity was achieved using a phosphorimager (Molecular Dynamics, Foster City,
CA).
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.
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Wortmannin inhibition- A 0.1 mM stock of wortmannin (Sigma, St. Louis, MO) was lo
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prepared in 10% DMSO. Wortmannin was incubated with 5 µg of nuclear extracts for 30 fro
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minutes on ice. Plasmid DNA end-joining experiments as previously described were ://w
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then performed at 14˚ C. .jb
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Immunodepletion- Extracts (50 µg) were incubated with antisera for 1 hour at 4º C on a b/
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rotary wheel. The extract/antibody mixture was added to 25 ul of protein A-Sepharose t o
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beads (Sigma, St. Louis, MO) and incubated with rotation for 3 hours at 4º C. The beads ril 1
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were removed by repeated centrifugation at 12,000 X g and the supernant was used for 19
western blot analysis and end-joining experiments.
Bacterial transformation assay- End-joining assays were carried out as described
above. After the 14 C(cid:176) incubation, the reaction mixture was incubated at 37 (cid:176)C with calf
intestinal alkaline phosphatase (Boehringer Mannheim) for 1 hour. Samples were then
extracted with phenol-chloroform, ethanol precipitated, and resuspended in 10 µl TE
buffer, pH 8.0. Electrocompetent Escherichia coli (strain DH10B) were then
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electroporated with 1 m l of recovered plasmid DNA using a Gibco BRL Gene Pulser at a
field strength of 2.44 kV/cm and plated on ampicillin-containing LB plates containing
IPTG and 5-bromo, 4-chloro, 3-indoyl -b D-galactoside (X-gal). 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 Perkin-Elmer automated DNA sequencer (Microchemical Facility,
University of Minnesota).
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DNA end-joining in Fanconi anemia fibroblast extracts
Results
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 mg of
extract was incubated for 12 hours at 14(cid:176) C with 1 mg 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 (CC), linear dimer (D),
and higher-molecular weight (HM) products when the reaction mixture was analyzed by
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agarose gel electrophoresis. In end-joining experiments performed with blunt-ended lo
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substrate, CC was the predominant product formed. When cohesive-ended substrates fro
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were used, CC, L, and HMW products were detected on the ethidium-bromide stained ://w
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gels. .jb
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Figure 1a shows the results of an end-joining experiment performed with blunt- by/
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ended substrate. Scanning laser densitometry was used to quantitate the bands, and the t o
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percentage of linear substrate that had been rejoined was determined. This analysis ril 1
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revealed that the normal extract rejoined 34% of the linearized substrate as compared to 9
3%, 7%, and 7% respectively, by the FA-A, FA-C, and FA-D extracts.
To confirm that the FA-A, FA-C, and FA-D extracts were deficient in DNA end-
joining, 2-3 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 7 normal fibroblast strains in addition to the normal
fibroblast strain used in Figure 1a (PD.715.F) and tested for end-joining activity. Figure
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