Supplementary Information
Methods
Proteins
Human Aprataxin was PCR amplified from a HeLa cDNA library (Invitrogen)
and cloned into pET41a to generate pET41a-APTX. Recombinant human
HISAprataxin
GST-
was expressed in E. coli BL21-CodonPlus RP cells at 30°C for 3
hrs following induction with 0.4 mM IPTG. Cells were lysed using Bugbuster
reagent (Novagen). Aprataxin was then purified by chromatography on NickelNTA-agarose (Qiagen) and Glutathione Sepharose (GE Healthcare). Where
indicated, the GST-HIS tags were removed by thrombin cleavage. The active
site mutant
GST-HISAprataxin
H260A
was generated using the QuickChange II
site-directed mutagenesis kit (Stratagene) and purified as described for the
wild-type protein.
Recombinant human DNA ligase III-XRCC1 complex was a gift from Dr
Tomas Lindahl1, DNA ligase IV-XRCC4 complex was purchased from
Trevigen, and T4 DNA ligase was from New England Biolabs.
S. cerevisiae HNT3, PCR amplified from yeast genomic DNA, was
cloned into pET11a-HIS-TEV2 using SacII and KpnI restriction sites.
HISHnt3
was expressed in E. coli BL21 (DE3) and purified using Nickel-NTA-agarose.
Genomically TAP-tagged3 Hnt1, Hnt2, Hnt3, Apa1 and Apa2 were
immunoprecipitated from yeast whole cell extracts using IgG-sepharose, in
buffer containing 40 mM HEPES, pH 7.5, 0.3 M potassium acetate, 4%
glycerol, 5 mM dithiothreitol, 0.1% NP-40, 5 mM NaF, 5 mM Na4P2O7 and a
cocktail of protease inhibitors (Roche) for 3 hours at 4°C. Immunocomplexes
were washed with DNA-adenylate hydrolysis reaction buffer. Proteins levels
were equalised following analysis by SDS-PAGE and western blotting using
peroxidase-anti-peroxidase soluble complex (Sigma).
Yeast cell-free extracts were prepared from logarithmically growing
1
cells by mechanical disruption using a freezer mill. Extracts were cleared by
ultracentrifugation and the soluble fraction dialysed against a buffer containing
50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM dithiothreitol.
APTX-defective chicken and human lymphoblastoid extracts
The generation of DT40 chicken B cells disrupted for Aptx will be described in
detail elsewhere. Briefly, a single round of targeting was sufficient for
disruption because the gene is located on chromosome Z (P.M.C. and K.W.C,
unpublished data). PCR analysis of the disrupted gene indicated a deletion
from Valine 78 onwards. Complemented Aptx-defective (and wild-type control)
cells were obtained by transfection of a pCMV vector encoding Myc-tagged
chicken Aprataxin, followed by G418 selection of stable clones. Single clones
of mutant and wild-type cells expressing equivalent amounts of
MYCAprataxin
were amplified and maintained for further analysis.
The human AOA1 lymphoblastoid cell lines Ap1 and Ap3 are described
elsewhere4. Whole cell extracts were prepared from human and chicken cells
as described5.
Aprataxin-targeted mice, primary cortical astrocytes, and astrocyte cell
extracts
The disruption of Aptx in described schematically in the legend to
Supplementary Figure S7a, and detailed characterisation of the mice will be
described elsewhere. In brief, the two Aptx exons encompassing the HIT
domain were constitutively deleted using an appropriate targeting construct,
creating a frame-shift that results in loss of the C-terminal 181 amino acids
including the histidine triad and zinc finger domains. Expression of the mutant
Aptx transcript was confirmed in various tissues, including brain, by northern
blot analysis and by genomic and RT-PCR.
2
Mouse cortical astrocytes were prepared from the brains of postnatal
day 4 wild type and Aptx-/- pups and maintained as monolayers in DMEM/F12
(1:1 mix) supplemented with 15% foetal calf serum, 2 mM L-glutamine, 100
U/ml penicillin, 100 µg/ml streptomycin, and 0.1 µg of mouse epidermal
growth factor (Sigma E1257). Extracts were prepared by lysing the astrocytes
in 20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 mM EGTA, 100 mM NaCl, 1%
Triton X-100, and a cocktail of protease inhibitors (Roche) for 30 min on ice.
Soluble material was recovered by centrifugation at 10,000 rpm for 5 min at
4°C. Protein concentrations were determined using the BioRad protein assay
kit with BSA as a standard.
Preparation of DNA-adenylates
The covalent DNA-adenylate intermediate was prepared essentially as
described6 and is indicated in the schematic of Fig. 1a. In brief, an 18-mer
(oligo 1: 5’-ATTCCGATAGTGACTACA-3’) was 5’-32P-labelled using T4
polynucleotide kinase (New England Biolabs) and 50 µCi [-32P]-ATP (GE
Healthcare) for 15 mins at 37°C followed by 15 mins chase using 0.5 mM
unlabelled ATP. The 18-mer was then annealed with a 36-mer (oligo 2: 5’TGTAGTCACTATCGGAATGAGGGCGACACGGATATG-3’) in the presence
of a third 18-mer oligonucleotide (oligo 3: 5’-CATATCCGTGTCGCCCTC-3’)
terminated with a 3’-dideoxy residue. All oligos were purchased from Sigma
and purified by denaturing gel electrophoresis and elution. For the annealing,
1 µg of oligos 1 and 3 were annealed with 2 µg oligo 2. The resulting nicked
duplex was purified by neutral PAGE and treated with 100 nM T4 DNA ligase
in ligation buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM DTT, 25 g/ml
bovine serum albumin, 1 mM ATP) overnight at room temperature. Since the
nick cannot be ligated due to the 3’-dideoxy, an abortive ligation reaction
takes place resulting in the adenylation of the 5’-terminus of oligo 1. The DNA
was then denatured and the adenylated 18-mer separated from other DNA
species by purification on a denaturing 10% PAGE in the presence of 7 M
3
32P-labelled
urea. Following gel extraction, the
adenylated 18-mer was
annealed with oligo 2 in the presence of oligo 3 (without the dideoxy group).
Ligation reactions
Reactions (5 µl) contained 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM DTT,
25 µg/ml bovine serum albumin, 1 mM ATP, and 30 nM ligaseIII/XRCC1, 100
nM ligase IV/XRCC4, or 100 nM T4 DNA ligase.
GST-HISAPTX
(30 nM) was
added where indicated. After 3 min incubation at 37C to generate enzymeAMP complexes,
32P-labelled
DNA-adenylate (DNA V shown in Fig. 1a) was
added to a final concentration of 1 µM. After further incubation for 2 min,
reactions were stopped by addition of formamide and heated for 3 min at
90C. Products were analysed by 10% denaturing PAGE and
32P-labelled
DNA products were detected by autoradiography.
Direct measurement of AMP release
DNA adenylates were prepared essentially as above, except that the 18-mer
was
32P-labelled
on the AMP group rather than at the 5’-phosphate. The
nicked substrate (containing a 3’-dideoxy group) was assembled and labelled
with radioactive AMP by treatment with T4 DNA ligase in the presence of 5
µCi [-32P]-ATP (GE Healthcare). The adenylated oligo was then purified and
annealed as described above to produce the DNA-[32P-AMP] substrate. This
nicked DNA (1 µM) was incubated with
GST-HISAprataxin
(30 nM) in hydrolysis
reaction buffer for 5 mins at room temperature. The release of
32P-labelled
AMP was detected by thin layer chromatography using Polygram CEL 300
PEI plates (Machery-Nagel) and 0.5 M LiCl/40 mM formic acid buffer.
Preparation of synthetic 5’-adenylated oxidative SSB substrates
To prepare an oligonucleotide duplex containing a single-strand break with a
1 nucleotide gap and 3’-phosphate and 5’-AMP termini, a 25-mer (5’4
GACATACTAACTTGAGCGAAACGGT-3’)
was
5’-32P-labelled
with
T4
polynucleotide kinase, re-purified, and annealed with a 43-mer (3’TAGGCAACTTCGGACGAAACTGTATGATTGAACTCGCTTTGCC-5’) and a
3’-phosphorylated 17-mer (5’-TCCGTTGAAGCCTGCTT-3’-P). The annealed
duplex was then incubated with T4 DNA ligase to adenylate the 5’-32Plabelled 25-mer. The adenylated 25-mer was re-purified from a 17%
denaturing PAGE gel and re-annealed with the 3’-phosphorylated 17-mer and
43-mer.
Reactive oxygen treatment
‘Dirty’ DNA breaks were produced by treating X174 RF I DNA (95 nM) with
10 µM H2O2, 0.1 mM FeCl3, 0.2 mM EDTA, 100 mM NaCl, and 1 mM NADH
for 30 min at room temperature. Reactions were stopped by addition of EDTA
(5 mM), and the products passed through a G25 spin column (Amersham
Pharmacia). The DNA was then treated with 30 nM DNA ligase III-XRCC1
complex and 50 µCi [-32P]-ATP in ligation buffer without ATP for 4 hr at
37°C. Abortive ligation at ‘dirty’ break sites resulted in the incorporation of
32P-
labelled AMP at 5’-termini. Reactions were stopped by addition of EDTA (40
mM), and unincorporated radioactivity removed by passage through a G25
spin column.
As shown schematically in Fig 4b, the preparation of supercoiled
X174 RF I DNA (sc) contains some nicked open circular DNA (oc). ROSinduced damage leads to multiple breaks and DNA relaxation. Abortive
ligation reactions result in the formation of adenylated 5’-termini. Closely
positioned single-stranded breaks produce some linearised DNA.
Removal of ROS-induced DNA-adenylates
ROS-induced DNA-adenylates (8.5 nM) were treated with purified
GST-HisAPTX
(20 nM), or yeast extract from wt and hnt3 strains (150 µg), or human
5
extracts from normal and AOA1 lymphoblastoid cell lines (Ap1 and Ap3, 20
µg). The removal of DNA-adenylates was monitored by agarose gel
electrophoresis.
Preparation of ligase-adenylate complex
To form DNA ligase-[32P-AMP] complexes, DNA ligase III-XRCC1 or T4 DNA
ligase (1 µM) were incubated with 1 µCi [-32P]-ATP (GE Healthcare) in 50
mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol, 25 µg/ml bovine
serum albumin. The self-adenylation reaction was carried out for 5 mins at
30°C, and stopped by addition of EDTA to 25 mM.
Sequence analyses
To create a phylogenetic tree of the HIT superfamily of proteins, orthologues
of human HINT1, FHIT, GALT, APTX, and DCPS, representing each
subfamily, were identified in mouse, bony fish, and yeast using pBlast. The
respective HIT domains were extracted and aligned using Clustal W multiple
sequence alignment program. A Neighbour-Joining tree was constructed and
analysed by a bootstrap test with 1000 replications.
6
Supplementary references
1.
Nash, R. A., Caldecott, K. W., Barnes, D. E. & Lindahl, T. XRCC1
protein interacts with one of two distinct forms of DNA ligase III.
Biochemistry 36, 5207-5211 (1997).
2.
Rass, U. & Kemper, B. Crp1p, a new cruciform DNA-binding protein in
the yeast Saccharomyces cerevisiae. J. Mol. Biol. 323, 685-700 (2002).
3.
Ghaemmaghami, S. et al. Global analysis of protein expression in
yeast. Nature 425, 737-741 (2003).
4.
Clements, P. M. et al. The ataxia-oculomotor apraxia 1 gene product
has a role distinct from ATM and interacts with the DNA strand break
repair proteins XRCC1 and XRCC4. DNA Repair 3, 1493-1502 (2004).
5.
Baumann, P. & West, S. C. DNA end-joining catalyzed by human cellfree extracts. Proc. Natl. Acad. Sci. U.S.A. 95, 14066-14070 (1998).
6.
Chiuman, W. & Li, Y. Making AppDNA using T4 DNA ligase. Bioorg.
Chem. 30, 332-349 (2002).
7
Supplementary Figure Legends
Figure S1. Reaction mechanism of DNA ligases.
The reaction mechanism for ATP-dependent DNA ligation involves the
formation of two adenylated complexes (highlighted). Step 1: Adenylated DNA
ligase is formed when the active site lysine reacts with ATP. The ATP is
cleaved to AMP and pyrophosphate leaving the adenylate residue linked to
the lysine in the active site of the enzyme. This step of the reaction is
reversible. Step 2: The activated AMP residue of the enzyme-adenylate
intermediate is transferred to the 5’-phosphate at the nick in double-stranded
DNA
to
generate
a
covalent
DNA-AMP
complex
with
a
5’-5’
phosphoanhydride bond. Step 3: Unadenylated ligase catalyses displacement
of the AMP through attack by the adjacent 3’-hydroxyl group on the
adenylated 5’-site. Phosphodiester bond formation seals the nick.
Figure S2. Purification of recombinant human Aprataxin.
a, Purification of recombinant human
GST-HISAprataxin.
Extracts from E. coli
harbouring vector pET41a::APTX before (lane 1) and after (lane 2) induction
of N-terminally GST- and His-tagged Aprataxin. Purification of
GST-HISAPTX
by
affinity chromatography using Nickel-NTA-agarose (lane 3) and Glutathionesepharose (lane 4). Fractions were analysed by SDS-PAGE and stained with
Coomassie blue. GST-HISAPTX is indicated.
b, Purification of recombinant human Aprataxin. Lane 1: Extract of E. coli cells
expressing
GST-HISAPTX.
Lane 2:
GST-HISAPTX
after affinity chromatography
using Nickel-NTA-agarose. Lane 3: Untagged APTX after thrombin-release
from Glutathione-agarose. Proteins were analysed by SDS-PAGE followed by
Coomassie blue staining.
8
Figure S3. Direct measurement of AMP release from the DNA-adenylate
intermediate.
Reactions (5 µl) contained DNA-adenylate (1 µM DNA), in which the substrate
32P-directly
on the AMP group (see methods). Following
GST-HISAPTX,
aliquots (1 µl) were analyzed by thin layer
was labelled with
incubation with
chromatography and the release of
32P-labelled
AMP was detected by
autoradiography (Lane 3). Lane 1: marker lane showing free
32P-labelled
AMP.
Figure S4. Analysis of Aprataxin on the DNA ligase-adenylate complex.
Reactions (10 µl) contained DNA ligase-[32P-AMP] complexes, prepared as
described in Methods, in the presence or absence of
GST-HISAPTX
(1 µM).
Following incubation for 10 min at 30°C, the products were analysed by SDSPAGE. DNA ligase-[32P-AMP] complexes were detected by autoradiography.
Figure S5. Phylogenetic tree showing that Aprataxin (APTX) forms a distinct
branch of the HIT superfamily.
The five subgroups of the HIT family are indicated. Each includes a human
representative (Hs, Homo sapiens) and orthologues from mouse (Mm, Mus
musculus), bony fish (Dr, Danio rerio; Tr, Takifugu rubripes), and yeast (Sc,
Saccharomyces cerevisiae).
Figure S6. Extracts from Aptx-disrupted DT40 cells exhibit reduced ligation
activity with the DNA adenylate substrate.
Reactions were carried out with the
32P-labelled
DNA-adenylate, using whole
cell extracts (20 µg of total protein) from normal or Aptx-disrupted DT40 cells,
and the same cells complemented with APTX cDNA. Reactions were carried
out with unadenylated or adenylated DNA
schematically.
9
substrates as indicated
Figure S7. Generation and characterization of Aptx-/- mice.
a. Scheme for inactivation of Aptx. A 15 kb KpnI genomic fragment was
isolated from a BAC containing the Aptx genomic locus, and oligomers
containing LoxP sites were introduced into an AgeI site, while a NeoTK
selection cassette flanked by LoxP sites was introduced ~5kb downstream
into an XbaI site. Genomic DNA encompassing exons encoding the HIT
domain were deleted via Cre recombinase excision in ES cells to generate a
mutant Aptx allele. The resulting mutant Aptx transcript does not contain the
exons encoding the HIT domain, and also results in an out-of-frame mutation
leading to a premature stop codon (asterisk) at amino acid 161 of Aptx
(Genbank: NP_079821).
b. Northern Blot analysis of Aptx expression. Analysis of Aptx mRNA
expression using Northern analysis from a variety of tissues including brain
(cerebellum and cerebral cortex) obtained from Aptx+/+, Aptx+/- or Aptx-/- mice
showed an absence of mutant Aptx transcript in all tissues examined from the
homozygous KO mouse. An abundant 1kb Aptx transcript is present in all
tissues, while tissue variation in other Aptx alternate splices occurs. The
Northern probe was a cDNA fragment containing the deleted exons indicated
in (a). An ethidium bromide-stained RNA gel shows equal RNA loading for all
genotypes and tissues examined; 18s and 28s RNA bands are indicated.
10