From the Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
Received for publication, January 23, 2001
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ABSTRACT |
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The excision of nucleotides from DNA 3' termini
is an important step in DNA replication, repair, and recombination
pathways to generate correctly base paired termini for subsequent
processing. The mammalian TREX1 and TREX2 proteins contain potent
3' The 3' The TREX1 and TREX2 genes encode two closely
related mammalian 3' The availability of the TREX gene sequences has made it
possible to generate recombinant proteins in sufficient quantities to
explore the biochemical properties of these mammalian 3' Materials--
[ Plasmid Construction--
The mouse Trex1 gene was
recovered from the EST GenBankTM accession number AA242227 by
PCR using the primers 5'-CTCAGGAGGTAAAAAAGCATGGGCTCACAGA-3' and
5'-TTGACTTACTGCCCAGGT-3' and cloned into the pOXO4 vector (32) to
generate the pTrex1-314 vector. In this vector, transcription of the
complete 314-amino acid Trex1 open reading frame is under control of
the T7 promoter. Translation is controlled by the ribosome-binding site
engineered into the sequence of the PCR primer at a position eight
nucleotides from the initiating ATG of the 942-nucleotide Trex1 gene. The human TREX2 gene was recovered
from the COSMID clone GenBankTM accession number AF002998 by PCR using
the primers 5'-TTCGGATCCTCTAGACCGGGTGCGGCACACTACATGTCCGAGGCACCCCG-3'
and 5'-ATTCAGGCCTCCAGGCT-3' and cloned into the pGEM-T Easy
Vector. The nucleotide sequence encoding the Genenase cleavage sequence
(Pro-Gly-Ala-Ala-His-Tyr) was engineered in-frame into the upstream
TREX2 PCR primer at a 5' adjacent position to the initiating
ATG of the TREX2 gene. The cloned TREX2 gene was
recovered from the pGEM vector by digestion with XbaI and
SalI and was ligated into the
XbaI/SalI-digested pMAL-c2 plasmid to generate a
pMAL-TREX2 vector. Expression from the pMAL-TREX2 vector generates
transcripts encoding fusion protein MBPTREX2. The position of the
Genenase recognition sequence permits cleavage of the fusion protein to
generate a 236-amino acid TREX2 protein. The sequences of the
pTrex1-314 and pMAL-TREX2 vectors were determined in both directions
using an automated DNA sequencer (PerkinElmer Life Sciences ABI Prism 377).
Overexpression and Purification of the Recombinant TREX
Proteins--
For overexpression of the Trex1 protein, pTrex1-314
plasmid was electroporated into E. coli BL21(DE3). Cells
were grown in LB (3 liters) at 25 °C to
A595 = 0.2, and
isopropyl-1-thio-
For overexpression of the MBPTREX2 fusion protein the pMAL-TREX2
plasmid was electroporated into E. coli XL1 Blue. Cells were grown in LB at 37 °C to A595 = 0.5, and
isopropyl-1-thio- Gel Filtration--
A Superdex 200 column was equilibrated in 20 mM Tris-HCl (pH 8.0), 2 mM dithiothreitol, 1 mM EDTA, 100 mM NaCl, 10% glycerol. The
protein mixtures (50 µg of each standard and Trex1 or TREX2) were
incubated at 4 °C for 30 min, and samples (200 µl) were applied to
the column at a flow rate of 0.5 ml/min. Fractions (250 µl) were
collected after a discarded volume of 9-10 ml. Samples of fractions
were concentrated in Microcons (Amicon), suspended in SDS sample
buffer, and separated by SDS-PAGE. Gels were stained with Coomassie
Brilliant Blue.
Exonuclease Assays--
The standard exonuclease reactions (10 µl) contained 20 mM Tris-HCl (pH 7.5), 5 mM
MgCl2, 2 mM dithiothreitol, 100 µg/ml BSA, 12.5 nM 5'-32P-labeled 23-mer oligonucleotide,
and TREX proteins (1-5 pg) as indicated in the figure and table
legends. Incubations were at 37 °C for the times indicated. For the
kinetic analysis a 5'-32P-labeled 21-mer or 37-mer:21-mer
partial duplex at concentrations that varied between 1 and 2000 nM and TREX proteins (5 pg) were used to generate initial
estimates of Km values. Reactions containing
substrate concentrations between 0.1 and 5 × Km values were performed for determination of the
kinetic constants. The exonuclease competition assays included heparin
(1 mg/ml) as a nonsubstrate inhibitor to compete with the various
partial duplex DNA substrates (1 nM) as described in the
figure legends. The amounts of Trex1 (100 pg) and TREX2 (1 ng) were
increased in these assays to detect 3' excision of the
5'-32P-labeled DNA substrates. Hybridization of oligomers
to generate partial duplexes has been described (33). Reactions were
quenched by addition of 30 µl of cold ethanol and dried in
vacuo. Pellets were resuspended in 7 µl of formamide, heated to
95 °C for 5 min, and separated on 23% denaturing polyacrylamide
gels. Radiolabeled bands were quantified using a PhosphorImager
(Molecular Dynamics). Linear regression and standard errors were
determined using SigmaPlot 5.0 (Jandel Corp.). All enzyme dilutions
were at 4 °C in 1 mg/ml BSA.
Protein Concentrations--
The protein concentrations were
determined by A280 using the following molar
extinction coefficients: Trex1, Purification of the Recombinant TREX Proteins--
The open
reading frames for the human and mouse TREX1 genes encode
proteins of 314 amino acids in length (16). Previous attempts to
overexpress the TREX1 protein in E. coli utilized gene
constructs encoding amino acids 11-314 and resulted in the recovery of
active TREX1 exonuclease in limited quantities (15, 31). Cloning of the
314-amino acid TREX1 open reading frame into a T7 RNA polymerase
expression vector has dramatically improved protein expression levels
using the mouse Trex1 gene, resulting in the purification of
~1 mg of Trex1 per liter of bacterial cell extract (Fig.
1). In contrast, the same expression
system has not improved yields of the recombinant human TREX1 protein
(data not shown). To generate the Trex1 protein, an E. coli
extract was prepared from induced cells containing the pTrex1-314
vector. Exonuclease assays demonstrate a 2000-fold greater activity in extracts prepared from induced cells containing the pTrex1-314 vector
relative to extracts prepared from cells containing the pOXO4
control plasmid. The Trex1 protein was purified by sequential chromatography using phosphocellulose, MonoQ, ssDNA cellulose, MonoS,
and Phenyl-Superose resins. Analysis of induced cell extracts by
SDS-PAGE does not reveal an obvious overproduction of the Trex1 protein
(Fig. 1, lane 2). Therefore, exonuclease assays were
performed to detect the Trex1 protein through the MonoQ chromatography
step, and SDS-PAGE was used to monitor purification in subsequent
steps. Analysis by SDS-PAGE of the pooled fractions from the MonoQ
column (Fig. 1, lane 4) reveals the presence of the
full-length Trex1 protein migrating to a position corresponding to 33 kDa and a fragment of Trex1 at the 30-kDa position. The identity of the Trex1 fragment was confirmed by N-terminal sequence analysis. The Trex1
fragment represents more than 50% of the overexpressed protein
recovered from the MonoQ column (Fig. 1, lane 4). The full-length Trex1 protein is enriched to greater than 75% in
subsequent steps by monitoring the purification using SDS-PAGE and
selectively pooling fractions containing the full-length Trex1 protein
(Fig. 1, lanes 4-7). This purified Trex1 protein was used
in subsequent experiments.
The human and mouse TREX2 genes encode a 236-amino acid
protein that has not been previously purified from an endogenous
source, nor had the recombinant TREX2 protein been produced. An
expression strategy was developed to produce a MBPTREX2 fusion protein
that could be cleaved using Genenase to permit purification of the 236-amino acid TREX2 protein (Fig. 2). An
E. coli extract was prepared from induced cells containing
the pMAL-TREX2 vector, and the MBPTREX2 fusion protein was
affinity-purified using an amylose resin (Fig. 2, lane 2).
The MBPTREX2 protein was incubated with Genenase in a time course
reaction to determine the optimal conditions for cleavage to generate
the MBP and the TREX2 protein (Fig. 2, lanes 3-6). Greater
than 90% cleavage of the fusion protein is obtained with minimal
degradation of the TREX2 protein. Chromatography of the cleaved fusion
protein using a MonoQ resin results in purification of the TREX2
protein with yields of ~0.5 mg of protein per liter of bacterial cell
extract (Fig. 2, lane 7). This purified TREX2 protein was
used in subsequent experiments.
Catalytic and Physical Properties of Trex1 and TREX2--
Several
experiments were performed to begin enzymatic and physical
characterization of the recombinant Trex1 and TREX2 proteins. The Trex1
and TREX2 proteins were examined for 3'
The gel filtration properties of Trex1 and TREX2 were examined to
determine the native structures of these proteins. Previous gel
filtration analysis of TREX1 purified from mammalian sources indicated
a native molecular mass consistent with a dimer structure (31, 35). To
confirm the dimer structure of Trex1 in the recombinant protein and to
determine the native structure for TREX2, these proteins were subjected
to gel filtration chromatography (Fig. 4). In separate experiments the
recombinant Trex1 or TREX2 proteins were mixed with protein standards
and subjected to gel filtration using a Superdex-200 column. The Trex1
protein elutes from the column coincident with the BSA standard,
indicating an apparent molecular mass of 67 kDa (Fig. 4A).
The calculated molecular mass of the recombinant Trex1 protein is
33,675 Da. These data support a dimer structure for the recombinant
Trex1 protein and provide evidence that the structure of the
recombinant Trex1 conforms to that of the native protein. The TREX2
protein demonstrates gel filtration properties indicating an apparent
molecular mass of 52 kDa (Fig. 4B). Because the calculated
molecular mass of TREX2 is 25,922 Da, a dimer structure is predicted.
The possible formation of heterodimers between these proteins has not
been tested.
Steady-state Kinetics of the TREX Proteins--
Our initial
experiments to measure the 3' Substrate Specificity of the TREX Proteins--
A competition
assay was developed to compare the 3' excision activities of Trex1 and
TREX2 using a series of alternative duplex DNA substrates. In this
exonuclease assay heparin was added as a nonsubstrate inhibitor to
compete with the 32P-labeled DNA substrate. The
nonsubstrate inhibitor heparin competes with the DNA substrate without
affecting the kcat and Km values for the DNA substrate (37). Therefore, the relative inhibitory effects of heparin on the 3' excision activities of Trex1 and TREX2
reflect the relative kcat/Km
values of the various duplex DNA substrates. This assay was used to
compare relative excision activities using various DNA substrates
without the necessity to determine kcat and
Km values for each DNA substrate. In previous work
it was shown that the Trex1 protein prefers mispaired 3' termini
several nucleotides in length within a partial duplex structure rather
than single-stranded DNA (15). The substrate preference for TREX2 has
not been previously described. Time course excision reactions using
partial duplex DNAs containing zero, one, or three mismatched
thymidines at the 3' terminus demonstrate the preference for mismatches
by Trex1 and also by TREX2 (Fig. 5). The
rates of excision of the 3' nucleotides were determined by quantifying
reaction products generated during the first 5 min of incubation. The
results demonstrate that the Trex1 protein has a 3-fold greater
activity on DNA containing one mispair and a 9-fold greater activity on
DNA containing three mispairs relative to DNA containing correctly base
paired 3' termini (Fig. 5A). Reaction products were allowed
to accumulate for 60 min to demonstrate the preference for the
mispaired 3' termini within the partial duplex DNA substrates. Using
the single mispaired DNA substrate, oligomer products 20 nucleotides in
length accumulate indicating the generation of the correctly paired 3'
termini. Similarly, using the DNA substrate containing three mispaired
nucleotides, oligomer products 18 nucleotides in length accumulate
indicating the generation of correctly paired 3' termini. The
accumulation of base paired 3' termini in these assays likely reflects
the change in kcat/Km
values for the substrate as the DNA structure is changed from a
mispaired to a paired partial duplex. When the same three DNA
substrates are incubated with the TREX2 enzyme, very similar results
are obtained (Fig. 5B). Using TREX2 a 3-fold greater
activity on DNA containing one mispair and a 15-fold greater activity
on DNA containing three mispairs relative to DNA containing correctly
base paired 3' termini is detected (Fig. 5B). Accumulation
of oligomer products corresponding to correctly base paired 3' termini
is apparent in reactions using the mispaired duplex substrates with
TREX2 after 60 min. These results demonstrate the similar substrate
preferences for multiply mispaired 3' termini by Trex1 and TREX2.
The single-stranded nature of the mispaired 3' terminus is not
sufficient to generate maximal excision rates by the Trex1 and TREX2
proteins. The rate of excision of the 3' nucleotide from a partial
duplex DNA containing three mispaired nucleotides is 3-fold greater
than the rate of excision of the 3' nucleotide from a single-stranded
oligomer, suggesting requirements for duplex DNA structure and
mispaired 3' termini (data not shown). These requirements are apparent
when measuring excision by Trex1 and TREX2 using duplex DNAs containing
3' overhangs (Fig. 6). Excision of the 3'
nucleotide was measured using Trex1 and TREX2 on duplex DNA with blunt
ends and on duplex DNA containing one or three nucleotides present as a
3' protruding end. The rate of excision for Trex1 using the
single-nucleotide 3' overhang was 11-fold higher than that for
blunt-ended DNA, and excision using the three-nucleotide 3' overhang
substrate was only 2-fold higher than that for the blunt-end duplex DNA
(Fig. 6A). The results for TREX2 demonstrate an 8-fold
higher excision rate for the single nucleotide 3' overhang relative to
the blunt-ended DNA and only 1.5-fold higher excision rate for the
three-nucleotide 3' overhang relative to the blunt-ended DNA (Fig.
6B). The rates of excision by Trex1 and TREX2 using the
blunt-ended DNA were the same as those using the partial duplex DNA
containing no mispaired nucleotides. In addition, excision rates for
Trex1 and TREX2 using the single nucleotide 3' overhang are 2- to
3-fold greater than those obtained using single-stranded oligomers
(data not shown). These results suggest a requirement for 3' mispairs
and for duplex DNA structure to generate maximal excision rates for
Trex1 and for TREX2.
A series of duplex DNAs was prepared to determine the substrate
requirements for single- or double-stranded DNA in the 5' overhang
region. An oligomer 12 nucleotides in length was hybridized to the
partial duplex DNA containing three mispaired 3' nucleotides to
generate a four-nucleotide gap and double-stranded DNA in the 5'
overhang region (Fig. 7). Excision rates
of mispaired 3' nucleotides by Trex1 (Fig. 7A) and by TREX2
(Fig. 7B) using this substrate were similar to rates
obtained using the partial duplex DNA containing three mispaired 3'
nucleotides with a single-stranded 5' overhang. However, when an
oligomer 16 nucleotides in length was hybridized to the partial duplex
DNA to completely eliminate the gapped region, a 9-fold decrease in the
excision rate was detected for both Trex1 (Fig. 7A) and for
TREX2 (Fig. 7B). This decreased excision rate is apparent in
the decreased accumulation of 21-mer after 60 min of incubation (Fig.
7, A and B). To more precisely determine the gap
length necessary for maximal excision by Trex1, a series of duplex DNA
substrates were prepared containing gaps between zero and four
nucleotides in length (Fig. 8). The Trex1
protein excises mispaired nucleotides in duplex DNAs containing gaps of
four, three, or two nucleotides at similar rates. A small decrease in rate (less than 2-fold) is detected in a duplex DNA containing a single
nucleotide gap, and an 8-fold decrease in rate is detected for the
removal of the second mispaired nucleotide when the gap is completely
eliminated. Similar results were obtained using TREX2 and these gapped
DNA duplexes (data not shown). These results indicate that a
two-nucleotide gap of single-stranded DNA in the 5' overhang region is
sufficient for maximal excision rates by Trex1 and TREX2.
The recombinant Trex1 and TREX2 proteins have been produced in
E. coli, and the purified proteins exhibit potent 3' An increasing number of mammalian genes have been identified that
encode proteins containing 3' Direct comparisons between the mammalian exonucleases have not been
performed, but the 3' excision activities detected in the recombinant
TREX proteins indicate a level of activity at least 1000-fold greater
than the 3' excision activities detected in the WRN, p53, hRAD1, hRAD9,
and MRE11 exonucleases. The catalytic properties of the TREX proteins
can be most closely compared with those of the bacterial exonucleases
contained in the epsilon subunit of DNA pol III (62), ExoI (63), ExoX
(21), and RNase T (29). The reported kcat values
for 3' nucleotide excision by these bacterial exonucleases range from
about 1 to 300 s The substrate specificity of the TREX proteins indicates that these
exonucleases could function in several DNA repair pathways by
generating DNA structures with correctly base paired 3' ends. The
preference of the TREX proteins for excision of mispaired 3' termini
might suggest a role in exonucleolytic proofreading. The mammalian DNA
polymerases The strategies presented in this study for the expression and
purification of the recombinant TREX proteins have allowed us to begin
characterization of the potent 3' excision activities of these enzymes.
A better understanding of the physiological role for these enzymes will
require additional biochemical and genetic studies. Availability of the
recombinant TREX proteins will facilitate further biochemical analysis
of these proteins and the identification of possible binding partners.
5' exonucleases capable of functioning in this capacity. To study
the activities of these exonucleases we have developed strategies to
express and purify the recombinant mouse Trex1 and human TREX2 proteins in Escherichia coli in quantities sufficient for
biochemical characterization. The Trex1 and TREX2 proteins are
homodimers that exhibit robust 3' excision activities with very similar
preferred reaction conditions and preferences for specific DNA
substrates. In a steady-state kinetic analysis, oligonucleotide
substrates were used to measure 3' nucleotide excision by Trex1 and
TREX2. The Michaelis constants derived from these data indicate similar
apparent kcat values of 22 s
1 for
Trex1 and 16 s
1 for TREX2 using single-stranded
oligonucleotides. The apparent KM values of 19 nM for Trex1 and 190 nM for TREX2 suggest relatively high affinities for DNA for both Trex1 and TREX2. An exonuclease competition assay was designed using heparin as a nonsubstrate inhibitor with a series of partial duplex DNAs to delineate the substrate structure preferences for 3' nucleotide excision by Trex1 and TREX2. The catalytic properties of the TREX proteins suggest roles for these enzymes in the 3' end-trimming processes necessary for producing correctly base paired 3' termini.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' exonucleases are frequently required in DNA repair
pathways to excise mismatched, modified, fragmented, or normal nucleotides from DNA 3' termini. The proteins containing 3'
5' exonuclease activity are in some cases large multiple-domain proteins, such as the proofreading exonucleases associated with many of the DNA
polymerases, and in other cases small single-domain proteins. Multiple
sequence alignments have been used with structural and mutagenesis
studies of the 3' exonuclease domains of Escherichia coli
DNA pol1 I and bacteriophage
T4 DNA pol to define the core sequence, structural, and functional
elements of an exonuclease domain (1-7). In these 3' exonucleases a
two-metal ion mechanism of nucleotide cleavage is indicated with four
negatively charged Asp or Glu residues and a Tyr residue located within
three conserved motifs, named ExoI, ExoII, and ExoIII. These
carboxylate residues in the Exo motifs identify DNA and RNA 3'
exonucleases that likely share a common catalytic mechanism for
nucleotide cleavage. A subset of the 3' exonucleases contains a His
rather than a Tyr in the ExoIII motif that is referred to as the
ExoIII
motif (8-10). The ExoIII
motif is characterized by the
presence of the sequence HXAXXD
rather than YXXXD and is detected in the
RNase T subfamily of exonucleases (1, 11-13). The structure of
E. coli ExoI suggests that the His in the ExoIII
motif
plays a role similar to the Tyr in the ExoIII motif of DNA pol I
(14).
5' exonucleases (15, 16). Analysis of the TREX
protein sequences using the COGNITOR program (available on the Web from NIH) suggests a possible relationship with proteins included in the "DnaQ" Cluster of Orthologous Groups of proteins (17). Although the functions of the TREX proteins are not known, the sequences of
these proteins suggest that these mammalian exonucleases most closely
relate structurally to the bacterial epsilon subunit of DNA pol III
(18), ExoI (19, 20), ExoX (21), and RNase T (22) enzymes. The epsilon
subunit of DNA pol III provides an exonucleolytic proofreading function
during DNA replication (18), and the ExoI and ExoX proteins have been
implicated in several DNA repair pathways, including mismatch, UV, base
excision, and recombination (21, 23-28). Earlier reports described the 3' ribonuclease activity of RNase T (22), but a more recent report
describes the potent 3' deoxyribonuclease activity of this protein
(29). Genetic experiments indicate that DNA repair defects resulting
from deficiencies in ExoI can be complemented by overexpression of
RNase T (30). Although direct evidence for functional roles in DNA
repair is not available for the TREX proteins, the ubiquitous expression of the TREX1 and TREX2 genes in human
cells supports a role for these 3' exonucleases in DNA repair pathways
(16). In a reconstituted base-excision repair system containing DNA pol
and DNA ligase IV-XRCC1, the accurate rejoining of a 3' mismatch
base at a single-strand break is dependent on addition of the TREX1
(also referred to as DNase III) protein (31).
5' exonucleases. We recently reported that the human and mouse
TREX1 gene encodes a protein of 314 amino acids in length
(16) and not 304 as previously indicated (15, 31). The human and mouse TREX2 gene encodes a protein of 236 amino acids in length.
To begin characterization of the TREX proteins we developed strategies to generate purified recombinant mouse
Trex12 and human TREX2
proteins. These proteins were used to measure the catalytic constants
that govern 3' nucleotide excision. A competition assay was designed to
permit a comparative analysis of 3' nucleotide excision using DNA
substrates with varying structures at the 3' termini. The results
presented here demonstrate the high catalytic efficiencies of the TREX
proteins that parallel the potent exonuclease activities detected in
some bacterial enzymes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, Superdex 200, MonoQ, MonoS, and Phenyl-Superose columns were from Amersham Pharmacia
Biotech. Phosphocellulose (P-11) was from Whatman. The heparin (H5027)
and ssDNA cellulose (D8273) were from Sigma-Aldrich Co. Amylose resin
and pMAL-c2 plasmid were from New England BioLabs. Oligonucleotides
were synthesized and purified in the Cancer Center of Wake Forest
University. T4 polynucleotide kinase and pGEM-T Easy Vector were from
Promega Corp. The mouse Trex1 EST (GenBankTM accession
number AA242227) was purchased from Research Genetics. The human
TREX2-containing COSMID (GenBankTM accession number
AF002998) was a generous gift from G. Nordsiek (Institute for Molecular
Biotechnology, Germany). The E. coli strain BL21(DE3)
(Novagen) and XL1-Blue (Stratagene) were used for protein expression.
-D-galactopyranoside was added to a
final concentration of 0.2 mM for an additional 12 h
at 25 °C. An extract was prepared by cell sonication in buffer A (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, and 10% glycerol), and the Trex1 protein was purified by
sequential chromatography using phosphocellulose, MonoQ, ssDNA
cellulose, MonoS, and Phenyl-Superose resins equilibrated in buffer A. The 3'
5' exonuclease activity was monitored upon elution from the
phosphocellulose and MonoQ columns using a standard exonuclease assay.
SDS-PAGE and Coomassie Blue staining were used to monitor subsequent
steps. A clarified extract was loaded onto a phosphocellulose column
(4.9 cm2 × 15 cm), and the column was washed with buffer A
and developed with a 200-ml linear gradient of 0-1000 mM
NaCl in buffer A. The peak of 3'
5' exonuclease activity eluted from
the column at 600 mM NaCl. The peak fractions were pooled,
dialyzed against buffer A, and loaded onto a MonoQ column 10/10. The
MonoQ column was washed and developed with a 100-ml linear gradient of
0-300 mM NaCl. The peak of 3'
5' exonuclease activity
eluted from the MonoQ column at 20 mM NaCl. Peak fractions
were pooled, dialyzed against buffer A, and loaded onto a ssDNA
cellulose column (2 cm2 × 5 cm). The column was washed,
and bound proteins were eluted with buffer A containing 500 mM NaCl. Eluted proteins were dialyzed against buffer A
loaded onto a MonoS column 10/10. The column was washed and developed
with a 100-ml linear gradient of 0-300 mM NaCl. The Trex1
protein eluted from the MonoS column at 20 mM NaCl.
Ammonium sulfate was added to pooled fractions to a final concentration
of 25% saturation. The sample was loaded onto a Phenyl-Superose column
10/10 equilibrated with buffer A containing 25% ammonium sulfate. The
column was washed with the equilibration buffer and developed with a
100-ml decreasing linear gradient of buffer A containing 25-0%
ammonium sulfate. The Trex1 protein eluted from the Phenyl-Superose
column at 15% ammonium sulfate. Fractions containing Trex1 were
pooled, and aliquots were stored at
80 °C.
-D-galactopyranoside was added to a
final concentration of 0.5 mM for an additional 4 h at
37 °C. A cell extract was prepared by sonication in buffer A, and
fusion protein MBPTREX2 was affinity-purified using an amylose resin as
described by the manufacturer. The 236-amino acid TREX2 protein was
cleaved from the MBPTREX2 fusion protein by incubation at 25 °C with
Genenase (1 mg of Genenase/100 mg of TREX2MBP). The cleavage reaction
was dialyzed against buffer A and loaded onto a MonoQ 10/10 column
equilibrated with buffer A. The MonoQ column was washed with 50 ml of
buffer A and developed with a 200-ml linear gradient of 0-500
mM NaCl. The TREX2 protein eluted at 180 mM
NaCl. Glycerol was added to the purified TREX2 protein to a final
concentration of 10%, and aliquots were stored at
80 °C.
= 25,110; MBPTREX2,
= 82,860; TREX2,
= 16,860 M
1
cm
1 (34).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of recombinant Trex1. The
Trex1 protein was purified from an extract prepared from pTrex1-314
plasmid-containing cells as described under "Experimental
Procedures." Samples of the extract (lane 2) and pooled
fractions from the phosphocellulose (lane 3), MonoQ
(lane 4), ssDNA cellulose (lane 5), MonoS
(lane 6), and Phenyl-Superose columns (lane 7)
were separated by SDS-PAGE. Lane 7 contains 2 µg of the
final Trex1 preparation, and lane 1 contains molecular
weight standards. The positions of migration for Trex1 and the
standards are indicated. The gel was stained with Coomassie Brilliant
Blue.
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Fig. 2.
Purification of recombinant TREX2. The
MBPTREX2 fusion protein was affinity-purified from a cell extract
prepared from pMAL-TREX2 plasmid-containing cells as described under
"Experimental Procedures." Cleavage of the TREX2 protein from the
MBPTREX2 fusion protein (lane 2) by Genenase was monitored
by SDS-PAGE of samples taken after 1 day (lane 3), 2 days
(lane 4), 3 days (lane 5), and 4 days (lane
6) of incubation. The cleaved TREX2 protein was subjected to MonoQ
chromatography to generate the final TREX2 preparation (lane
7). The positions of migration of the MBPTREX2, MBP, TREX2, and
molecular weight standards are indicated.
5' exonuclease activity using
a single-stranded oligomer to confirm the presence of this activity in
the recombinant proteins and to establish the relative 3' nucleotide
excision rates for these enzymes (Fig. 3). Incubation of increased amounts of
Trex1 (Fig. 3, lanes 1-5) or TREX2 (Fig. 3, lanes
6-10) results in degradation of a 23-mer at similar rates. The
picogram quantities of enzyme required to detect the 3'
5'
exonuclease activity of Trex1 are consistent with previous estimates of
the activity for this enzyme purified from mammalian cells (35, 36).
These results also indicate similar catalytic efficiencies for Trex1
and TREX2. An analysis of reaction requirements was performed to
establish optimal conditions for Trex1 and TREX2 3'
5' exonuclease
activities (Table I). These results
demonstrate that very similar conditions are required for Trex1 and
TREX2. Both enzymes prefer the Mg2+ divalent cation with
maximal activity detected at a concentration of 5 mM. Both
enzymes can utilize Mn2+ with decreased activities
detected, and neither enzyme can utilize Zn2+ for
catalysis. The Trex1 and TREX2 activities are inhibited by NaCl or KCl
at concentrations above 50 mM, are stabilized by the addition of at least 0.1 mg/ml BSA, and exhibit pH optima in the range
of 7.5-8.0 (Table I). The similar reaction conditions required for
Trex1 and TREX2 likely reflect similar catalytic mechanisms.
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Fig. 3.
The 3' excision activities of the recombinant
Trex1 and TREX2 proteins. Standard exonuclease reactions were
prepared with the 32P-labeled 23-mer as described under
"Experimental Procedures." Dilutions of Trex1 or TREX2 were
prepared at 10 times the final concentrations, and samples (1 µl)
containing 0.5 pg (lanes 2 and 7), 1.0 pg
(lanes 3 and 8), 2.0 pg (lanes 4 and
9), and 5.0 pg (lanes 5 and 10) were
added to reactions. No enzyme was added to the reactions in
lane 1 or lane 6. Reaction
products were subjected to electrophoresis on a 23% polyacrylamide
denaturing gel. The position of migration of the 23-mer is
indicated.
Reaction requirements for Trex1 and TREX2
5' exonuclease reactions were prepared using 5 pg of
Trex1 or TREX2 and the indicated reaction modification. Incubations
were 5 min for Trex1 and 10 min for TREX2. Products were separated on
polyacrylamide gels and quantified.
View larger version (96K):
[in a new window]
Fig. 4.
Gel filtration analysis of Trex1 and
TREX2. Mixtures containing protein standards (b = BSA, 67 kDa; o = Ovalbumin, 48 kDa; c = chymotrypsinogen A, 25 kDa; and r = RNase A, 14 kDa)
and Trex1 (A) or TREX2 (B) were prepared and
subject to gel filtration as described under "Experimental
Procedures." Samples (50 µl) of the indicated fractions were
subjected to 12% SDS-PAGE, and gels were stained with Coomassie
Brilliant Blue. The positions of migration of Trex1, TREX2, and the
protein standards are indicated.
5' exonuclease activities of the
recombinant Trex1 and TREX2 proteins indicated similar, but not
identical, excision properties. A steady-state kinetic analysis was
performed to more precisely quantify the catalytic constants governing
excision of 3' nucleotides by these enzymes (Table
II). Excision by the recombinant Trex1
and TREX2 proteins was measured using varied concentrations of a
single-stranded 21-mer to determine the catalytic rate constant
kcat and the apparent dissociation constant
Km without consideration for DNA structure. Excision
by Trex1 and TREX2 was also measured using varied concentrations of a
double-stranded DNA substrate prepared by hybridizing the 21-mer to a
37-mer generating a partial duplex with a 16 nucleotide 5' overhang.
The kcat values determined for Trex1 and TREX2
from these data reveal the high turnover numbers for these enzymes
during excision of 3' nucleotides (Table II). These
kcat values vary by less than 2-fold when
measured using either the single- or double-stranded DNA substrates. In
contrast, the Km values are 10- and 23-fold lower
for Trex1 than for TREX2 using the single- and double-stranded DNA
substrates, respectively. The similar kcat
values and the disparate Km values suggest
differences in the equilibrium binding constants that control
dissociation of Trex1 and TREX2 from DNA. The Km values of 19 nM for single-stranded DNA and 15 nM for double-stranded DNA determined for Trex1 indicate
that this exonuclease has a relatively high affinity for the DNA
substrate.
Steady-state kinetic analysis of Trex1 and TREX2
View larger version (30K):
[in a new window]
Fig. 5.
Trex1 and TREX2 prefer mispaired 3' termini
in a partial duplex DNA. Exonuclease competition reactions (50 µl) were prepared containing a partial duplex with a paired 3'
terminus (lanes 1-5), with one mispair (lanes
6-10), and with three mispairs (lanes 11-15) at the
3' terminus and the nonsubstrate inhibitor heparin as described under
"Experimental Procedures." The Trex1 (A) or TREX2
(B) was added, and samples (10 µl) were removed at the
indicated times after incubation. The positions of migration of the
oligomers are indicated.
View larger version (38K):
[in a new window]
Fig. 6.
Excision of 3' nucleotide overhangs by Trex1
and TREX2. Exonuclease competition reactions (50 µl) were
prepared containing a partial duplex with a blunt-ended 3' terminus
(lanes 1-5), with one 3' protruding nucleotide (lanes
6-10), and with three 3' protruding nucleotides (lanes
11-15) and the substrate inhibitor heparin as described under
"Experimental Procedures." The Trex1 (A) or TREX2
(B) was added, and samples (10 µl) were removed at the
indicated times after incubation. The positions of migration of the
oligomers are indicated.
View larger version (30K):
[in a new window]
Fig. 7.
The effect of duplex DNA in the 5' overhang
on excision of 3'-mispaired termini by Trex1 and TREX2.
Exonuclease competition reactions (50 µl) were prepared containing a
partial duplex with three mispairs and a single-stranded 5' overhang
(lanes 1-5), a four-nucleotide gap in the 5' overhang
(lanes 6-10), and a duplex structure in the 5' overhang
(lanes 11-15) and the nonsubstrate inhibitor heparin as
described under "Experimental Procedures." The Trex1 (A)
or TREX2 (B) was added, and samples (10 µl) were removed
at the indicated times after incubation. The positions of migration of
the oligomers are indicated.
View larger version (16K):
[in a new window]
Fig. 8.
The effect of gap length on excision of
3'-mispaired termini by Trex1. Exonuclease competition reactions
(50 µl) were prepared containing a partial duplex with three mispairs
and a 5' overhang containing a four-nucleotide gap (lanes
1-5), a three-nucleotide gap (lanes 6-10), a
two-nucleotide gap (lanes 11-15), a one-nucleotide gap
(lanes 16-20), and no gap (lanes 21-25) and the
nonsubstrate inhibitor heparin as described under "Experimental
Procedures." The Trex1 was added, and samples (10 µl) were removed
at the indicated times after incubation. The positions of migration of
the oligomers are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5'
exonuclease activities. The TREX1 gene was initially
identified by sequencing peptides from a protein detected as the most
prevalent 3' excision activity in mammalian cell extracts (15, 31, 36).
The limited quantities of enzyme recovered from endogenous mammalian
tissues suggest that TREX1 contains a catalytically robust 3'
5'
exonuclease, and the measured activity in the recombinant Trex1 protein
confirms this observation. In our standard exonuclease reactions 5 pg
(0.15 fmol) of Trex1 degrades more than 95% of a 23-mer to products less than 10 nucleotides in length (Fig. 3). The
kcat and Km values for Trex1
determined in the kinetic analysis (Table II) indicate a
kcat/Km value of 1.2 × 109 M
1 s
1 as a
lower limit for the apparent second-order rate constant for substrate
binding. This value approaches the theoretical diffusion-controlled encounter frequency of an enzyme with its substrate (38). To our
knowledge the TREX2 protein has not been purified from an endogenous
mammalian tissue. The TREX2 gene was identified in data base
searches using the TREX1 gene as a query sequence (15, 31).
The properties of the recombinant TREX2 protein also demonstrate a
potent 3'
5' exonuclease with kcat values
similar to those of Trex1. The 10-fold higher Km
value for DNA measured with TREX2 relative to Trex1 indicates a lower
affinity for DNA. The nature of the apparent difference in DNA binding
affinity is not known.
5' exonuclease activities. In addition
to TREX, these genes include Werner syndrome
(WRN) (39-41), p53 (42), hRAD1
(Ustilago maydis REC1) (43, 44), hRAD9
(45), and hMRE11 (46, 47). Genetic defects have not yet been
identified in the TREX genes, but defects in the other exonuclease genes indicate functions in various pathways of DNA replication, repair, and recombination for this diverse collection of
proteins. A conserved nuclease domain was identified in the N-terminal
region of the WRN protein (48, 49), and biochemical studies confirm the
presence of the 3'
5' exonuclease activity in an N-terminal fragment
containing this domain (50). Genetic defects in the WRN protein
increase genomic instability (51). The multifunctional p53 protein
contains a fold structure similar to E. coli ExoIII and the
APEX protein (52-54). The 3' excision activity has been localized to
the central core, sequence-specific DNA binding domain that functions
in cell-cycle checkpoint control (42, 55). The sequence of the MRE11
protein indicates there is a relationship to the "SbcD" DNA
repair exonuclease family of proteins (56). Genetic and biochemical
analysis of MRE11 supports a role for this exonuclease in double-strand
break repair (46, 47, 57, 58). There are no obvious sequence
relationships between hRAD1 (REC1) or hRAD9 and any known exonucleases,
but there is a possible structural relationship between hRAD1 (REC1) and the PCNA sliding clamp family of proteins (59). Defects in these
DNA damage checkpoint response proteins affect DNA repair pathways that
are required for genomic stability (60, 61).
1. These turnover numbers are comparable
to the kcat values of about 20 s
1
determined for Trex1 and TREX2 (Table II). The apparent
Km values for DNA substrates for ExoI, ExoX, and
RNase T are between 1 and 12 nM compared with
Km values of 19 and 190 nM for Trex1 and
TREX2, respectively. The Trex1 and TREX2 proteins exhibit a
distributive pattern of excision similar to the ExoX and RNase T and
not the highly processive excision properties characteristic of the
ExoI enzyme. The dimeric structures of the recombinant Trex1 and TREX2
proteins suggest a structural relationship with the RNase T protein.
The RNase T is a dimer, and dimerization is required for activity (64).
It is not known if dimerization of the TREX proteins is required for
activity. The 236-amino acid TREX2 protein is 45% identical to the
N-terminal region of the 314-amino acid TREX1 protein. Therefore the
dimeric structures of Trex1 and TREX2 suggest that amino acids involved
in the subunit interface likely reside within the first 236 amino acids
of the TREX proteins. Perhaps the C-terminal region of TREX1 provides a
unique region for this exonuclease to interact specifically with
additional proteins.
and
do not contain proofreading exonucleases. The
TREX proteins could remove nucleotides misinserted by these DNA
polymerases to generate the paired 3' termini necessary for continued
DNA synthesis. The TREX1 protein functioning as an editing exonuclease
for the DNA pol
has been demonstrated in a reconstituted base
excision repair assay (31). During strand-specific mismatch repair in
human cells, the excision step is exonucleolytic, requiring a 3'
5'
exonuclease when the strand break is positioned 3' to the mismatch
(65). The activity of the TREX proteins in conjunction with a helicase
activity could provide the necessary components to facilitate the
excision step in DNA mismatch repair in human cells as has been
indicated for ExoI in bacteria. The structural relationship between
ExoI, ExoX, and RNase T with the TREX proteins makes it tempting to
speculate on a potential role for the TREX proteins in UV excision
repair. Genetic studies support the participation of ExoI, ExoX, and
RNase T in UV and mismatch repair (21, 23, 30). Finally, DNA
recombinational pathways involved in double-strand break repair often
require a 3'
5' exonuclease to remove nucleotides from 3' termini.
The TREX proteins could function to eliminate unpaired 3' ends to
permit subsequent ligation.
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ACKNOWLEDGEMENT |
---|
We thank Scott E. Harvey for excellent technical assistance throughout this work.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA75350 and CA12197.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.
To whom correspondence should be addressed: Dept. of Biochemistry,
Wake Forest University School of Medicine, Winston-Salem, NC 27157. Tel.: 336-716-4349; Fax: 336-716-7200; E-mail: fperrino@wfubmc.edu.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M100623200
2 The murine orthologs of TREX have the approved symbols of Trex1 and Trex2.
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ABBREVIATIONS |
---|
The abbreviations used are: pol, polymerase; ExoI, E. coli exonuclease I; ExoX, E. coli exonuclease X; TREX, Three prime Repair EXonuclease; WRN, Werner syndrome protein; APEX, APurinic/apyrimidinic EXonuclease; PCR, polymerase chain reaction; EST, expressed sequence tags; MBP, maltose binding protein; ssDNA, single-strand DNA; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.
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