From the Department of Cell and Tumor Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010 and the § Division of Biology, California Institute of Technology, Pasadena, California 91125
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ABSTRACT |
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Yeast exonuclease 1 (Exo1) is induced during
meiosis and plays an important role in DNA homologous recombination and
mismatch correction pathways. The human homolog, an 803-amino acid
protein, shares 55% similarity to the yeast Exo1. In this report, we
show that the enzyme functionally complements Saccharomyces
cerevisiae Exo1 in recombination of direct repeat DNA fragments,
UV resistance, and mutation avoidance by in vivo assays.
Furthermore, the human enzyme suppresses the conditional lethality of a
rad27 Mutations cause genomic instability and gene dysfunction, many of
which affect cell growth and lead to tumorigenesis. Fortunately, in
normal cells, mutation rate is low due to the existence of different
DNA repair systems such as mismatch, excision, and recombinational DNA
repair pathways. In humans, DNA mutation accumulation is a critical
step in carcinogenesis. Dysfunctional mutations of DNA mismatch repair
genes such as MSH2, MLH1, and PMS2,
are the main cause of the hereditary non-polyposis colorectal cancer
(1-6). A portion of sporadic cancers is due to acquired mutations in mismatch repair genes as well (7). Mutations of genes encoding nucleotide excision repair proteins including XPG nuclease are linked
to xeroderma pigmentosum (8-17).
Nucleases play important roles in several pathways including DNA
replication, repair, and recombination. DNA fragments containing a
lesion are removed by the combined efforts of a helicase and a
nuclease. For instance, in the DNA mismatch repair of E. coli, exonucleases, exo I (3'-5' excision) and exo VII or Rec J
(5'-3' excision) are responsible for the bi-directional removal of DNA fragments containing mismatched lesions (18, 19). For DNA recombination
or repair of double strand DNA breaks through recombination, an
important step is to generate a 3' single-stranded terminus for strand
invasion. This step is accomplished by 5'-3' exonucleases (20-22).
During DNA replication the removal of RNA primers in the lagging strand
also requires 5'-3' nuclease activity (23). In E. coli and
other bacteria, the removal of RNA primers is performed by the 5'-3'
exonuclease activity of DNA polymerase I (24-27). The polA
ex1 mutant, defective in 5'-3' exonuclease activity, exhibits
retarded joining of nascent DNA fragments. In eukaryotes, DNA
polymerases lack an intrinsic 5'-nuclease. Removal of RNA primers is
carried out by an independent enzyme, Rad27/FEN-1 nuclease, with both
flap endonuclease and 5'-3'exonuclease activities (28-38). Disruption
of the flap endonuclease gene RAD27 in the yeast
Saccharomyces cerevisiae resulted in DNA replication
defective symptom including the conditional lethality (39-42).
Survival of the null mutant at 30 °C suggests that other enzymes
with 5'-3' exonuclease activity could back up the function of
Rad27/FEN-1 in DNA replication (29, 43).
A 5'-3' exonuclease, called exonuclease 1 (Exo1) in
Schizosaccharomyces pombe and S. cerevisiae and Tosca in Drosophila melanogaster, has recently been identified and partially characterized (44-48). The
enzyme is a non-processive double-stranded DNA nuclease (44). Both the
messenger RNA level and enzyme activity were dramatically induced
during meiosis in S. pombe (44, 45). In D. melanogaster, the gene is specifically expressed in the early
embryogenesis and female germline (46). The S. cerevisiae
and human exonucleases 1 interact with mismatch repair protein Msh2 as
demonstrated by the two-hybrid system and immuno-coprecipitation
(48-50). Disruption of EXO1 increased mutation rate in both
S. pombe and S. cerevisiae cells, indicating that
EXO1 is a mutator gene (45, 48). Detailed analysis of the
mutational spectrum of the exo1 Human and S. cerevisiae exonuclease sequences have high
similarity. This similarity leads us to predict that functions of these
two enzymes are also similar and the human gene may complement the Exo1
functions in yeast. In addition, when Szankasi and Smith (45)
identified the S. pombe gene (SpEXO1), they
noticed that the encoded protein displays significant similarity to the
proteins of rad13 (XPG/Rad2) and rad2 (FEN-1/Rad27) nuclease families. However, the similarities were limited to regions of N-terminal putative nuclease domains and the major part at C terminus was largely
divergent (45). We hypothesize that the human exonuclease 1 may further
complement the Rad27 nuclease functions in RNA primer removal during
lagging strand DNA synthesis based on the observations of the sequence
conservation between these two proteins, their enzymological
properties, and phenotypical characteristics (i.e. conditional lethality) of the rad27 null mutants. Our in
vitro and in vivo data demonstrate that the human
exonuclease can functionally complement the yeast homologues (yExo 1 and yRad27) in recombination, UV resistance, RNA primer removal during
the Okazaki fragment processing, and mutation avoidance.
Materials--
A cDNA clone (number 843301) harboring a
putative human exonuclease 1 was obtained from ATCC (Manassas, VA).
Oligonucleotides used in this study were synthesized in the City of
Hope Cancer Center core facility. The vector pET-28a and E. coli strains BL21 and BL21 (DE3) were from Novagen (Madison, WI)
and E. coli strain XL2 blue and pBSK vector were from
Stratagene (La Jolla, CA). Yeast expression vector pDB20 was a gift
from D. Becker, California Institute of Technology. Restriction enzymes
were obtained from New England Biolabs (Beverly, MA). Yeast culture
media including YPD, synthetic complete (SC), minimal sporulation, and
synthetic dextrose minimal (SD) media were prepared according to
Sherman et al. (53). Amino acids and all other medium
components and chemicals were purchased from Sigma. Isopropyl
Yeast Gene Disruption--
All of the yeast strains constructed
and used in this study are listed in Table
I. For construction of the
EXO1 null mutant strain, the EXO1 gene was
amplified by PCR1 using
primers with HindIII and NcoI sites (exolpl
(AGAAAGCCATGGGTATCCAAGGT) and exolp2
(CTCCGAAAGCTTTCAGTGATGATGGTGGTGGTGTTTACCTTTATAAACAAATTGGGAA) and cloned
into the vector pET-28b (Novagen). The marker gene URA3 was
obtained from plasmid YEp24 and inserted into PstI sites, replacing the EXO1 coding sequence between positions 170 and
828. The fragment containing the
exo1::URA3 construct was removed from the plasmid with restriction enzymes XbaI and
NotI. The exo1::URA3 fragment was then
transformed into yeast strains W1021-7c and W1089-6c (Table I).
Ura3+ transformants were analyzed by PCR using primers
exolp3 (CGAACAAACTGAAAGGCGTAG) and exolp4 (GTCTTGAGGCATTTCGACGAG) and
Southern blotting to verify disruption of EXO1. The
exo1 rad51 double mutant was obtained by crossing
exo1 (FDAB15D) with rad51 (U687, a gift from Adam Bailis, City of Hope). RAD27 gene was deleted by
transforming the wild type strains W1021-7c and W1089-6c with a
PCR-generated DNA fragment. The PCR product was generated using YEp13
containing LEU2 marker gene as a template and two primers
KNRTH1
(CATCGATGAAAAGCGTTGACAGCATACATTGGAAAGAAATAGGAAACGGACACCGGAAGTTAACTGTGGGAATACTCA) and KNRTH2
(AGCTGTTCCTTTGTCTTAGGCACCACTTGGAAGAACCCATCTAACCTACCCTGACTACGTCGTAAGGCCGTTT) (underlined sequences were designed to amplify LEU2 marker
gene, the remaining sequences are from RAD27). Another pair
of the primers was used to confirm the deletion of RAD27
gene by PCR: RAD27F (AAACGCGACGCGTAACATCG) and RAD27R
(ATATGCCAAGGTGAAGGACC). Null mutant strains of RAD51 and
RAD51/EXO1 were from laboratory stock (51).
Complementation of ScEXO1 and HEX1 in S. cerevisiae--
Two
vectors, pDB20 and pRS314, were used for the expression of
EXO1 in S. cerevisiae. pDB20 is a URA3
and ADH1 promoter-based yeast expression vector. Two
plasmids, PDB-ScE and PDB-hE, were constructed to express
Sc- and h- exonuclease 1 in S. cerevisiae. PDB-ScE has an insertion of S. cerevisiae EXO1
(ScEXO1) at the HindIII site of pDB20 while
PDB-hE has an insertion of human EXO1 (hEXO1) at
the NotI site of pDB20. pRS314 is a yeast subclone vector
using TRP 1 as a selection marker. PRS-ScE and PRS-hE were constructed
to complement the exonuclease 1 function in the
rad27::URA3 background. PRS-ScE has an insertion
of Padh-ScEXO1-Tadh (Padh, ADH 1 promoter; Tadh,
ADH1 terminator) at SacI and ApaI sites. PRS-hE
has an insertion of Padh-hEXO1-Tadh at BamHI site. The plasmids were transformed into different S. cerevisiae
strains for exonuclease 1 functional complementation as listed in Table I.
Overexpression and Purification of hExo1--
Full-length human
EXO1 coding sequence was subcloned into pET-28a vector
(Novagen) using the cDNA clone harboring hEXO1 and PCR
primers 5exo1 and 3exo1 containing NheI and SalI
sites (5exo1, GACTGTGCTAGCATGGGGATACAGGGATTG and 3exo1,
TGTCACTGTCGACAATCCAAAGTTTTTCCAG), respectively, yielding pET-HEX. Both
pET-28a and pET-HEX were transformed into BL21 (DE3) cells (Novagen).
Colonies were inoculated into 50 ml of LB broth supplemented with 30 µg/ml kanamycin and cultured overnight. The culture was pelleted and
transferred in 4 × 1-liter of sorbitol medium (54). Cultures were
grown at 37 °C to OD 0.6 followed by induction at 25 °C with 1 mM isopropyl- Nuclease Activity Assay--
To prepare substrates for
ribonuclease activity assays, an upstream primer
(5'-TTTTATAACTCGGAGAGCCGTAATG-3', the bold nucleotides are
the 5' single-stranded portion of exonuclease 1 substrates) and a
5'-labeled 21-ribonucleotide/30-deoxyribonucleotide junction oligo
(5'-gggaacaaaagcuugcaugccTGCAGGTCGACTCTAGAGGATCCCCGGGTA-3', the
lowercase indicates the ribonucleotides) were annealed to the 76-mer
template
(5'-TTTTTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTTTGTTCCCCATTACGGCTCTCCGAGTTAT-3') resulting in a Okazaki fragment-like substrate (ribo-substrate-1). To
test the ability of human exonuclease 1 in removal of the last ribonucleotide attached to DNA, 5'-labeled
mono-ribonucleotide/30-deoxynucleotide junction oligo
(5'cTGCAGGTCGACTCTAGAGGATCCCCGGGTA-3') were annealed to a 55-mer
complementary strand
(5'-TTTTTACCCGGGGATCCTCTAGAGTCGACCTGCAGTAGACGTCTGACACAGCCGT-3') to form a nicked duplex substrate (ribo-substrate-2). An upstream primer (5'-TTTTACGGCTGTGTCAGACGTCTA-3') has also been added to the above substrate (ribo-substrate-2) to form the
ribo-substrate-3. Furthermore, a 5' end-labeled oligo
(5'-ACGGCTGTGTCAGACGTCTACTGAGGTCGACTCTAGAGGATCCCCGGGTA-3') was annealed
to the template strand of the ribo-substrate-2 to form blunt end
double-stranded DNA as a substrate for a 5' DNA exonuclease activity
assay. Based on the property that hExo 1 has a negligible activity on a
single-stranded DNA, all unlabeled 5' ends of the substrates have four
additional nucleotides to form the single-stranded ends and
block the enzyme activity. The ribonuclease reactions were performed as
described previously (55). Briefly, a standard reaction mixture
contained 0.8 pmol of [ Recombination Assay--
The recombination assay was based on a
construct of URA3 gene flanked at both ends by the same DNA
fragment (413 bp) from the HIS3 gene direct repeats (see
Fig. 2 and Ref. 56). The construct was inserted into the
HIS3 gene in the wild type S. cerevisiae cells.
These cells were hybridized with exo1 or rad27
null mutants. They were then sporulated and dissected to produce the
exo1 or rad27 null mutant strains with the assay construct.
The strains were cultured in the SD-URA medium to saturation. They were
then plated onto histidine minus SD (SD-HIS) medium. The optimal
concentration for plating was tested based on three different
concentrations (5 × concentrated, saturated, and 10 × diluted). For the wild type and exo1 null mutants, 100 µl
of saturated culture cells was optimal and plated onto SD-HIS medium.
After 3-4 days, HIS3+ recombinant colonies were counted
and recombination rate was calculated by the number of recombinants
divided by the total number of viable cells.
UV Sensitivity Analysis--
Analysis was carried out as
described previously (51) except that the strains in this study were
transformed with plasmids, pRS314, pRS-ScE, or pRS-hE, respectively
(Table I). Strains were grown in tryptophan minus SD (SD-TRP) liquid to
saturation at 30 °C and cell density was measured with a
spectrophotometer (600 nm), diluted in water, and plated on SD-TRP agar
plate. The plates were exposed to different UV dosages (254 nm
germicidal lamp) and incubated in the dark for 3-4 days at 30 °C
before colonies were counted. Survival rate was determined based on the
ratio between colony counts with and without UV treatment. Survival curves represent the average from at least six independent experiments.
Suppression Analysis--
The pDB overexpression vector with or
without insertion of either HEX1 or ScEXO1 was
transformed into the rad27 null mutant and wild type strain
of S. cerevisiae. To observe colony growth, the
transformants were streaked onto the SD-URA medium side by side with
duplications. They were incubated at 37 and 30 °C, respectively. The
transformants were also cultured in the SD-URA liquid medium to
saturation to measure the growth curve. The saturated cultures were
then normalized and inoculated in 10 ml of SD-URA liquid medium. The
growth was determined using a spectrophotometer in different time
intervals for the curve.
Mutator Phenotype Analysis--
To observe the role of
EXO1 in mutation avoidance, the mutator phenotype of
exo1 deletion mutant (exo1) and the ones
transformed with ScEXO1 and HEX1 were determined
using an assay based on cycloheximide resistance (57). The strains were
cultured to saturation in 2.5 ml of SD-TRP liquid medium. For control,
0.2 µl of the culture was diluted by 1 ml of distilled water and 50 µl of the diluted samples was plated onto the SD-TRP agar plate for
cell number counting. The remaining samples were spun down and plated
onto the SD-TRP agar plate with 10 µg/liter cycloheximide. Mutation rate for each strain was calculated from six independent experiments.
Protein Sequence Comparison Predicted That Human Exonuclease-1 Is a
Yeast Functional Homolog--
A cDNA clone (number 843301)
harboring a putative human exonuclease 1 was obtained from ATCC.
Sequence of cloned cDNA was determined in our laboratory and
deposited in GenBank (accession number AF060479) and translated into
the protein sequence. Sequence of S. cerevisiae exonuclease
1 was obtained from the NCBI protein data base. They were then aligned
using Optimal Global Alignment of Two Sequences at EERIE (Nimes,
France) (Fig. 1). Overall sequence
identity and similarity between human and S. cerevisiae
EXO1s are 27 and 55%, respectively. The identity and similarity
between the putative nuclease domains of the yeast Exo1 and Rad27
nuclease in are 28 and 57%. High sequence conservation between the
human and S. cerevisiae predicts that they are functional homologs and leads us to test in yeast if the human enzyme can complement the characterized functions of the yeast enzyme.
Expression of HEX1 Restores the Recombination Rate in an exo1 Expression of HEX1 Ablates the UV Sensitivity of an exo1 Purified hExo1 Efficiently Removes RNA Primers from Okazaki
Fragment Model Substrates--
Full-length human exonucleases-1
cDNA was cloned into the pET28a vector and expressed in E. coli using sorbitol medium as described previously (54, 58). PCR
primers were designed to add both N- and C-terminal His tags onto the
recombinant protein. The final products of fast protein liquid
chromatography purification using a nickel affinity column contained
two protein bands (approximately 93 and 45 kDa). Microsequencing of
both polypeptides revealed the expected N-terminal sequence of the
human exonuclease 1 deduced from its cDNA sequence (MGIQGLL).
Therefore, the smaller polypeptide is a C-terminal degradation product
of the 93-kDa protein. All of the Exo1 homologs (S. cerevisiae, S. pombe, and human) possess a 5'-3'
double-stranded DNA exonuclease activity. To explore the possibility
that the enzyme might participate in RNA primer removal during
eukaryotic DNA replication lagging strand DNA synthesis, three Okazaki
fragment model substrates were prepared as ribo-substrates-1, -2, and
-3 (Fig. 4) that mimic dynamic lagging
strand DNA replication intermediates. Indeed, the enzyme possesses a 5'
riboexonuclease activity on all of the substrates. A blunt ended
double-stranded DNA duplex was included as a control (Fig. 4,
A-D). The time course of the enzyme reaction indicates that
the reactive efficiency of the enzyme with ribo-substrate-1, -2, and
double-stranded DNA substrate are similar (Fig.
5, A-D). hExo1 digests both
RNA and DNA with a duplex substrate with or without an upstream primer. However, the enzyme releases mononucleotides with the substrate-1, -2, and DNA duplex while it cleaves mono-, di-, and trinucleotides simultaneously from the 5' end with substrate-3 (an upstream primer was
added to the substrate-2, Fig. 4C).
Expression of Both ScEXO1 and HEX1 Suppressed the Lethality of
rad27 Expression of Both ScEXO1 and HEX1 Reversed the Mutator
Phenotypes--
Due to the functions of the EXO1 gene in
recombinational mismatch correction and probably in RNA primer removal,
deletion of the gene increased yeast mutation rates 29-fold in the
cycloheximide resistance assay (Table
III). The rate increased about 6-fold in the CANr and HOM assays (59). Expression of both
ScEXO1 and HEX1 reversed the mutator phenotypes
of an exo1 null mutant and the mutation rate was reduced to
the wild type level. These data indicate that the human gene is a
functional homolog of the yeast gene and can play a role in mutation
avoidance in heterogeneous cellular environment.
Sequence and in Vitro Functional Conservation of Eukaryotic
5'-Exonuclease 1--
The enzyme has so far been identified in
S. pombe, S. cerevisiae, D. melanogaster, and human (44-52). Sequence similarity of Exo1s
from these eukaryotic organisms indicates evolutionary and functional
conservation of this important group of 5'-exonucleases. The
biochemical properties of hExo1 are similar to that of
ScExo1. The enzyme is a non-processive 5'-3' double-stranded
and single-stranded DNA exonuclease. In S. cerevisiae, the
5'-3' exonuclease activity is 2-fold more on double-stranded DNA than
on single-stranded DNA. The purified protein from S. pombe,
however, has very low 5'-3' single-stranded DNA exonuclease activity.
It is more specific for the double-stranded DNA. It is possible that
the truncated protein purified from S. pombe lacks a
structure responsible for 5'-3' single strand DNA exonuclease activity.
Resembling the ScExo1, human exonuclease 1 has both
activities but prefers a double-stranded DNA substrate to
single-stranded DNA. However, it does not discriminate between RNA and
DNA (Fig. 4 and see below). Because the observed sequence conservation
of the putative nuclease domains between the exonuclease-1 and flap
endonuclease-1 (see Refs. 28, 29, 60, and 62 for additional information
on FEN-1 nuclease), we tested the flap endonuclease activities of human
exonuclease-1 with a typical flap DNA substrate with DNA or RNA as the
single-stranded portion and human flap endonuclease-1 as a positive
control. The result indicates that the enzyme does not possess a
specific flap endonuclease activity. A detailed description of the
human exonuclease 1 substrate specificity will be published elsewhere.
Human Exonuclease 1 Functionally Complements the Yeast DNA
Recombination and UV Resistance and Mutation Avoidance--
Expression
of human exonuclease 1 in yeast complements several characterized
phenotypes in the exo1
In S. cerevisiae, it has been shown that Exo1 is involved in
UV DNA damage repair, which is distinct from nucleotide excision repair
pathway (49). In this study, we revealed that hEXO1
complements the function of ScExo1 in UV DNA damage repair,
indicating that hExo1 could be involved in UV DNA damage repair in
human cells as well. Among the different UV DNA damage repair pathways,
the XPG/Rad2 nucleotide excision repair pathway is a major pathway. In
humans, dysfunction of this repair pathway results in xeroderma pigmentosum syndrome. Epistatic analysis in yeast showed that the UV
DNA damage repair involving Exo1 is independent of XPG/Rad2 nucleotide
excision repair and other UV DNA damage repair pathways (51). The minor
UV sensitivity is due to the failure of DNA replication-driven bypass
compensation mechanism.
Failure of mismatch correction during DNA recombinational repair
increases mutation rate. Deletion of ScEXO1 caused a 29-fold increase in cycloheximide resistance in S. cerevisiae. The
assay is based on the fact that mutation in the yeast gene
CYH2 can lead to resistance to cycloheximide, an inhibitor
of eukaryotic protein synthesis. The gene product of CYH2 is
ribosomal protein L29, a component of the 60 S ribosomal subunit. In
most cases, resistance to cycloheximide is due to a transversion
mutation resulting in replacement of a glutamine by a glutamic acid in position 37 of Leu-29 (57). Mutation rates in a ScEXO1
deletion mutants assayed with another system such as CANr
and HOM3 are only increased 6-fold compared with wild type (59). The
different mutation rates determined may be due to the following reasons: 1) the CANr assay is a forward assay but not
particularly sensitive to the point mutations such as transversions; 2)
the HOM3 assay is sensitive to single base deletions and additions,
which happens frequently in mismatch repair defective cells. This is a
reversion assay. The mutant strain causes at a high rate the deletion
of a single T in a run of 7 Ts. Otherwise, the HOM3 protein does not
tolerate much sequence variation elsewhere. Table III also shows that
HEX1 complements the function of ScExo1 and
reverses the mutator phenotype in exo1 Human Exonuclease 1 Backs Up the Function of Flap Endonuclease 1 in
DNA Replication RNA Primer Removal--
Fig. 4 shows a
time-dependent ribonuclease activity of hExo1 on RNA/DNA
hybrid duplexed to a DNA template regardless of multiple or
monoribonucleotide proceeding the DNA portion. This activity may
indicate the involvement of Exo1 in RNA primer removal during lagging
strand DNA synthesis. In prokaryotes, the role of RNA primer removal
during DNA lagging strand synthesis is played by the 5'-3' exonuclease
activity of DNA polymerase I (24-27). In eukaryotes, the function of
removing RNA primers of the lagging DNA strand is performed by the
FEN-1/Rad27 nuclease in two alternative pathways. One pathway is that
RNase H removes all ribonucleotides except for the last one adjunct to
DNA fragments; then, the last ribonucleotide is removed by FEN-1/Rad27
nuclease. The second pathway is performed by FEN-1/Rad27 nuclease via
its flap endonuclease activity independent of RNase H. If there are
only these two pathways involved in the removal of the RNA primers,
disruption of the FEN-1/RAD27 gene in yeast
should completely block the RNA primer removal pathways and the null
mutant may be lethal. However, disruption of ScRAD27 caused
a conditional lethality phenotype (39-42): the null mutant cells could
still grow at the normal temperature (30 °C) but become lethal at
37 °C. For this reason, a third pathway for removing RNA primers has
been proposed (29) and Exo1 is one of the best candidates for this
pathway due to its 5'-3' exonuclease activity. The hExo1 was as
efficient in removal of ribonucleotides as deoxynucleotides, resembling
the function of Rad27 in RNA primer removal pathway in our experiments.
As we have also shown, the overexpression of HEX1 as well as
ScEXO1 in yeast S. cerevisiae cells suppressed
the temperature-sensitive phenotype of rad27/fen-1 null mutant at
37 °C. This result indicates that the Exo1 enzyme is involved in
removal of RNA primers during lagging strand DNA synthesis. Bambara's
hypothesis (29) on the candidate nucleases for initiator RNA removal is
supported by the fact that overexpression of EXO1 suppresses
the temperature-sensitive phenotype of rad27/fen-1 null mutant at
37 °C and the lethality of the double knockout of RAD27
and EXO1 (48).
Recently, however, it has also been proposed that the unligated Okazaki
fragments of the rad27 null mutant can also be repaired by
the recombinational double strand break repair pathway (43). This
hypothesis is based on the fact the disruption of
FEN-1/RAD27 can elevate recombination rate by
20-30-fold (43).2 In
addition, the double knockout of RAD27 and RAD51
or RAD52 (the key protein components of double strand break
repair) is lethal. The double strand break repair pathway requires
5'-3' exonuclease to generate the single-stranded 3'-overhangs for
strand invasion. EXO1 is a good candidate for this process
as well. Meanwhile, if EXO1 is the only enzyme so far
available, which possibly generates the single-stranded 3'-overhangs in
a eukaryotic cell, deletion of EXO1 would block the double
strand DNA break repair pathway. The double knockout of
RAD27 and EXO1 would result in the cell lethality
as that of RAD27 and RAD51 or RAD52.
This hypothesis is supported by the fact that the knockout of
EXO1 reduced recombination rate and the double knockout of
RAD27 and EXO1 is indeed lethal. However, this
hypothesis does not explain the result that the overexpression of
EXO1 suppressed the temperature-sensitive phenotype of rad27
null mutant unless the overexpression of Exo1 facilitates an
alternative process for the RNA primer removal. With the above information in mind, we propose that Exo1 might be involved in two
pathways relevant to DNA replication. On one hand, it could back up the
function of FEN-1 to remove RNA primers; on the other hand, it is
involved in the double strand DNA break repair pathway to generate
3'-overhang, correct the mismatches, and remove unligated Okazaki
fragments in the rad27 mutant.
mutant, symptomatic of defective RNA primer removal. The
purified recombinant enzyme not only displays 5'-3' double strand DNA
exonuclease activity, but also shows an RNase H activity. This result
indicates a back-up function of exonuclease 1 to flap endonuclease-1 in
RNA primer removal during lagging strand DNA synthesis.
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ABSTRACT
INTRODUCTION
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cells suggested a role
for the encoded protein in mismatch correction, most likely during
homologous recombination. In addition, deletion of yEXO1 significantly decreased both meiotic and mitotic recombination rates
(45, 47, 48). One speculation is that the nuclease plays a role
creating 3' single-stranded complementary tails, thereby promoting
joint molecule formation. Moreover, the null mutant displays a minor UV
sensitivity unlikely due to a nucleotide excision repair deficiency but
may be due to a defective DNA replication by-pass pathway (51). These
findings indicate that the Exo1 homologs function as 5'-3' exonucleases
in mutation prevention via multiple DNA metabolic pathways. More
recently, human homolog of the exonuclease 1 has been identified and
the gene was named HEX1 (52). It is specifically expressed
in fetal liver and adult bone marrow, suggesting that the enzyme may
operate prominently in processes specific to hemopoietic stem cell
development. The gene has been mapped to 1q43, a region lost in some
cases of acute leukemia and in several solid tumors (49, 52).
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-D-thiogalactoside was purchased from Roche Molecular
Biochemicals (Indianapolis, IN).
S. cerevisiae strains used in the study
-D-thiogalactoside for 4 h. The cells were harvested and stored at
80 °C until use. All of
the purification steps were performed at 4 °C. The cells was thawed
in a mixture of ice and water and resuspended in buffer A (20 mM Na2HPO4, pH 7.9, 0.5 NaCl) with
10 mM imidazole. Cells were sonicated and the lysate was
then centrifuged at 17,000 rpm for 45 min to remove cell debris. A 5-ml
HiTrap Chelating Ni2+ column was equilibrated with buffer A
using Fast Protein Liquid Chromatography system (Pharmacia). After
loading, the column was washed with 25 ml of buffer A with 10 mM imidazole, 25 ml of buffer A with 60 mM
imidazole, and then eluted with a 50-ml linear gradient from 60 to 500 mM imidazole in buffer A at 2 ml/min. The fractions were
run on the 10% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue R-250 to visualize the purity of protein. Buffers of the
fractions were exchanged to 10 mM Tris, 150 mM
NaCl, pH 8.0, by HiTrap desalting column (Pharmacia). Protein
concentrations were determined using the Bio-Rad protein assay.
-32P]nucleic acid
substrate, 50 mM Tris-HCl, pH 8.0, 10 mM
MgCl2, and 10 ng of enzyme in 13 µl and were incubated at
33 °C for 30 min. The labeled substrates were prepared in a
concentration ratio of 1 labeled oligonucleotide to
103-106 unlabeled oligonucleotide. The
reactions were stopped by adding an equal volume of stop solution
(U. S. Biochemical Corp.), mixed, boiled for 3 min, and cooled in
ice. 3 µl of each reaction product was run on a 15% denaturing
polyacrylamide gel and exposed to Kodak x-ray film for an image. For
time-dependent kinetic analysis, the cleavage reaction was
stopped in different time points. The products were resolved on a
denaturing gel. Quantification of the band intensities was done with a
PhosphorImager (Stratagene, CA) and the computer program IPLab Gel
assay (Signal Analytics, VA).
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ABSTRACT
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Fig. 1.
Sequence alignment of hExo1,
ScExo1, and the putative nuclease domain of
ScRad27. | indicates identical amino acids; : indicates
similar amino acids; and * the conserved amino acids composing a
nuclease active site as demonstrated in crystallography and
site-directed mutagenic experiments of homologous enzymes of FEN-1 (61,
62). Overall sequence identity and similarity between human and
S. cerevisiae EXO1s are 27 and 55%, respectively.
Underlined are the conserved regions between exonuclease 1 and Rad27 nuclease.
Strain--
ScEXO1 is involved in spontaneous mitotic and meiotic
recombination between direct repeats (45, 47). Complementation of this
function during S. cerevisiae mitotic growth by
HEX1 was determined by measuring the rate of recombination
between nontandem direct repeats of the HIS3 genes (Fig.
2). The two HIS3 alleles are
truncated at their 3' and 5' ends, respectively, and are separated by
plasmid vector sequences including the URA3 gene. The end
repeats of the HIS3 gene in this system are 413 bp in
length. Strains containing this duplication form can grow on the SD
medium without uracil. Recombination events that excise the DNA between
the repeats and restore the HIS3 gene are visualized as the
recombinant cells can grow on the medium without histidine.
Recombination rate in a strain containing this direct repeat and a
disruption of the EXO1 gene were 58% lower than that of the
wild type strain hosting the HIS3 gene construct as a
control. Expression of either ScEXO1 or HEX1
restored the recombination rate to the wild type level as shown in
Table II.
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Fig. 2.
A recombination assay used in this
study. The yeast strain, HUWT (wild type) and HUWT1
(exo1 ), containing a recombination substrate on the
chromosome that consists of two nontandem direct repeats of truncated
alleles (415 bp) of the his3 gene separated by the
URA3 sequence were used in this experiment. They were
transformed with three plasmids: PRS314, PRS-ScE, and PRS-hE to measure
the complementation ability of HEX1 in recombination (see
the results in Table II).
His+ recombination frequency in strains containing the
his2-3'::URA3::his3-5'
Strain--
Deletion of the EXO1 gene in S. cerevisiae causes a minor UV sensitivity (51). Epistatic analysis
indicated that the observed phenotype was not due to the defective
RAD2-dependent nucleotide excision repair or
loss of its known function in RAD51-dependent double strand break-induced recombination pathway or mismatch repair
pathway. It is most likely due to its involvement in a DNA replication
by-pass pathway. Double deletion of EXO1 and
RAD51 made the strain very sensitive to UV treatment.
Introduction of the human and yeast EXO1 expression plasmids
into this double deletion mutant strain recovered the UV sensitivity
close to the level of the rad51
mutant (Fig.
3).
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Fig. 3.
UV sensitivity of S. cerevisiae
exo1 null mutatnt is reversed by the expression of
Sc- or h-EXO1. As revealed previously (51),
the single deletion of S. cerevisiae EXO1 only exhibits a
minor UV sensitivity whereas the double deletion of EXO1 and
RAD51 has a synergistic effect. For this reason, the double
deletion mutant was used to test if the HEX1 could
complement the function of S. cerevisiae EXO1 in the UV DNA
damage repair. , AB15Be (wt/pRS 314);
, AB1Ba (exo1
/pRS314);
, U687a (rad51
/pRS314);
, FDERa (exo1
rad51
/pRS314);
,
FDERb (exo1
rad51
/PRS-ScE);
, FDERc (exo1
rad51
/PRS-hE).
The data was averaged from six independent experiments.
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Fig. 4.
The purified Exo1 protein shows the 5'-3'
double strand exonuclease activity and 5'-3' riboexonuclease
activity. Exonuclease activity of human recombinant Exo1 was
tested with a blunt ended DNA duplex substrate and illustrated in
panel D. The 5'-3' riboexonuclease activities were tested
with Okazaki fragment-like substrates and illustrated in panels
A-C. Sequence of oligoes used to make the substrates is given in
the text. The assays were performed as described under "Experimental
Procedures." Time points were 0, 1, 1.5, 2.5, 5, 7.5, 10, and 20 min.
Lanes 1-8, triangles denote increasing reaction
time. Dots in the substrate scheme represent RNA portion.
The filled dots indicate the 5' single strand blockage to
the 5' Exo1 activity. Arrows with nt (nucleotide)
show the length of the substrates and products; * indicates the
radioactively labeling position.
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Fig. 5.
Human exonuclease 1-driven reactions
with three different RNA-DNA/DNA and DNA/DNA duplex substrates.
The reactions have been conducted under the conditions described in the
legend to Fig. 4 and under "Experimental Procedures" with excessive
substrate to the enzyme. 103-106-fold more of
unlabeled oligoes than the labeled oligo were used to construct the
substrates. Densitometry was used to quantify the conversion of
substrate to product. A, reaction with blunt ended
double-stranded DNA substrate; B, reaction with RNA-DNA/DNA
substrate (ribo-substrate-1); C, reaction with
monoribonucleotide-DNA/DNA substrate (ribo-substrate-2); D,
superimposition of A, B, and C. The data is from
the average of three independent experiments.
Mutant at 37 °C--
Yeast Rad27 nuclease and its
mammalian homolog FEN-1 functions in RNA primer removal during lagging
strand DNA synthesis (29, 38, 43). rad27 null mutants
displayed a conditional lethality phenotype: the cells grow slowly at
30 °C but were arrested in S-phase at 37 °C. The mutants also
have a hyper-recombination phenotype. This is indicative of long-lived
regions of single-stranded DNA in the chromosome and is symptomatic of
a defect in Okazaki fragment processing. A partial defect in this
process indicates that an inefficient nuclease backs up the function of
the Rad27 nuclease. Exonuclease-1 shares a conserved nuclease domain
with the Rad27 nuclease (Fig. 1). Deletion of both EXO1 and
RAD27 in S. cerevisiae leads to complete
lethality of cells (data not shown). Expression of ScEXO1
and HEX1 in a rad27 null mutant promotes the
colony formation at 37 °C even though the human gene has less capacity to remove RNA primers in yeast as the transformants formed smaller colonies (Fig. 6). All of these
strains were grown in the liquid YPD medium at 37 °C to determine
the growth curve. The results indicated that the expression of both
Sc- and human EXO1 gene made the rad27 null
mutant grow more closely to the wild type even though the strain
harboring the human EXO1 gene grew more slowly than the
rad27
mutant with the ScEXO1 (Fig. 7).
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Fig. 6.
Overexpression of Sc- or
h-Exols suppresses the lethality of RAD27/FEN-1 mutant at
37 °C. Wild type (wt) and rad27 mutant (IC2) strains were grown
on the SD-URA plate with and without the introduction of Sc-
and h-EXO1s. a, AB15Ba (wt/pDB20); b,
AB15Bb (wt/PDB-ScE); c, AB15Bc
(wt/PDB-hE); d, IC2-1c (rad27/PDB-hE);
e, IC2-1b (rad27 /PDB-ScE); f, IC2-1a
(rad27
/PDB20).
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Fig. 7.
Cell growth curve of rad27 mutant strain with
and without the introduction of Sc- and
hEXOls. Cells were grown in SD-URA liquid medium
at 37 °C starting with the same amount of initial culture. Samples
were taken in every 2-3-h time intervals to measure the OD units. The
data were averaged from three independent experiments. , AB15Ba
(wt/pDB20);
, AB15Bb (wt/PDB-ScE);
, AB15Bc
(wt/PDB-hE); *, IC2-1a (rad27
/PDB20);
, IC2-1b
(rad27
/PDB-ScE); ×, IC2-1c (rad27/PDB-hE).
Expression of hEXO1 reverses cycloheximide resistance of the exo1 null
Saccharomyces cerevisiae
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutants. hExo1 complements the
function of ScExo1 in recombination (Table II). The
recombinational rate decreases 2-fold when the EXO1 gene was
deleted in our assay system, where the repeat fragments were about 400 bp. 7-fold decrease was reported when the repeats were extended to 900 bp (45). It was also demonstrated in vitro that the enzyme
significantly promotes the recombination of the two DNA fragments with
end overlapping sequence. As revealed in S. pombe, mRNA
level and enzyme activity was induced during meiosis (44, 45) and in
D. melanogaster, it is only expressed in the early
embryogenesis of the female germline (46). We speculate that the
functional 5'-exonuclease 1 may be involved in recombinational mismatch
correction during meiosis in mammalian cells as no such eukaryotic
enzyme has been identified to date.
mutants. This
result implies that hExo1 could play a role in the mutation avoidance
mechanism in human cells. The result is also consistent with the role
of exo1 in mismatch correction in S. pombe (44).
The protein interacts with Msh2 (48, 49) and double knockout of
EXO1 and MSH2 showed the similar mutation rate as
the single knockout of MSH2. Altogether, the evidence available indicates that these two proteins may work together in a
mutation avoidance mechanism.
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ACKNOWLEDGEMENT |
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We thank Dr. Adam Bailis for stimulating discussions and suggesting the use of the CYH2 assay system and Tim O'Connor, Sharon Lin, and Geoffrey Frank for critical reading of the manuscript and technical assistance.
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FOOTNOTES |
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* This work was supported by a National Institutes of Health, NCI Grant CA82468 (to B. H. S.).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.
Present address: Dept. of Biology, Stanford University, Stanford,
CA 94305.
¶ To whom correspondences should be addressed. Tel.: 626-301-8879; Fax: 626-301-8972; E-mail: bshen{at}coh.org.
2 J. Qiu and B. Shen, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s).
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REFERENCES |
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