From the Laboratory of Molecular Genetics, NIEHS,
National Institutes of Health, North Carolina 27709, ¶ Department of Biochemistry and Molecular Biophysics, Washington
University School of Medicine, St. Louis, Missouri 63110, and
Lindenwood University, St. Charles, Missouri 63301
Received for publication, September 24, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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To address the different functions of Pol Efficient and faithful maturation of Okazaki fragments during DNA
replication in eukaryotes depends on a coordinated degradation of the
RNA primer strand by one or more nucleases along with gap-filling DNA
synthesis by a replicative DNA polymerase followed by ligation of the
remaining nick. Previous models based on a combination of biochemical
and genetic studies have indicated a role for the flap 5'-endonuclease
FEN1 and the nuclease/helicase Dna2 in carrying out degradation
including the removal of a displaced flap and a role for DNA polymerase
Many DNA polymerases have an intrinsic 3'-5' exonuclease activity,
which corrects polymerase errors and prevents mutations. Recently, we
provided genetic evidence for the action of the 3'-5'-exonuclease of
Pol Okazaki fragment maturation is mediated by the concerted strand
displacement of Pol In this paper, we describe studies that indicate that the exonuclease
activity of Pol Materials and Strains--
The strains used in this study, wild
type, rad27-p, rad27-null, pol3
The 88-mer double hairpin oligonucleotide
(5'-C9AAAACCAACCCACT5GTGGGTTGGTTTTGGGA8CTTCTCCTTTCTCTCCT5GGAGAGAAAGGAGAAG-3')
was extended by a single dTMP residue with carrier-free
[ DNA Polymerase and Exonuclease Assays--
DNA polymerase assays
were carried out on DNase I-activated salmon sperm DNA (11). The
50-µl nuclease assay contained 20 mM Tris-HCl, pH 7.8, 8 mM MgAc2, 0.2 mg/ml bovine serum albumin, 4% glycerol, 1 mM dithiothreitol, 100 fmol of 3'-end-labeled pUC19 DNA,
and enzyme. The DNA substrate was prepared by linearizing pUC19 DNA
with EcoRI and filling in with dATP and
[3H]dTTP followed by purification of the DNA. Assays were
assembled on ice in Microfuge tubes and incubated at 37 °C
for 15 min. They were stopped by the addition of 100 µl of 25 mM EDTA, 25 mM sodium pyrophosphate, and 50 µg/ml carrier DNA followed by 125 µl of 10% trichloroacetic acid.
After 10 min on ice, the tubes were spun in a Microfuge for 10 min. 200 µl of the supernatant was added to a water miscible scintillation
fluid and counted in a liquid scintillation counter. All other
replication and Okazaki fragment maturation assays were essentially as
described by Ayyagari et al. (3).
Replication Assays--
Standard 30-µl assays contained 20 mM Tris-HCl 7.8, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 8 mM MgAc2, 1 mM ATP, 100 µM of each dNTPs, 100 mM NaCl, 100 fmol of primed template, 400 fmol (for
oligos) or 10 pmol (for SKII DNA) of RPA, and 150 fmol of all other
enzymes (RFC, Pol Yeast Genetic Methods--
Yeast genetic methods were as
described previously (4, 5). For the study of methylmethane sulfonate
(MMS) sensitivity, the double mutants pol3-5DV rad27-p were
obtained from a double mutant strain carrying pLC80
(RAD27-TRP1) as fresh plasmid loss isolates.
Colonies of 12 independent isolates were suspended in H2O
at a density of ~107 cells/ml. Serial 10-fold dilutions
were made in microtiter plates, and small (1 ul) drops of cells were
placed on the YPD and YPD + 4 mM MMS media.
To study the rescue of synthetic lethality by overexpression of
DNA2, plasmid pGAL18-DNA2 or vector pGAL18 was
introduced into the triple mutant strain pol3-5DV rad27-p
rad51-null harboring plasmid pLC80 (RAD27-TRP1). Such
triple mutants require the presence of plasmid pLC80 for growth (5).
Cells were grown on galactose medium lacking uracil (Gal-Ura) to allow
the loss of plasmid pLC80 under conditions of overexpressing
DNA2. Several independent isolates were picked and diluted,
and ~200 cells were plated on Gal-Ura medium. Three days later, the
cells were replica-plated on Gal-Ura and Gal-Ura-Trp. The
percent of colonies that were unable to grow on Gal-Ura-Trp was determined.
Two exonuclease-deficient forms of Pol Deficiency in Pol Characterization of Exonuclease-deficient Pol Exonuclease-deficient Pol
To be able to assess the contribution of the replication clamp PCNA to
strand displacement synthesis, we used a model oligonucleotide system
with terminal biotin-streptavidin anchors to prevent PCNA from sliding
off the DNA (18-20). In this system, displacement synthesis of a
downstream primer by wild-type Pol
Even in the presence of PCNA, strand displacement synthesis is preceded
by extensive pausing of the polymerase at the downstream primer. With
Pol
The model oligonucleotide system conveniently measures whether a
polymerase carries out strand displacement or not but does not
distinguish between initiation and elongation events. Elongation of
strand displacement can more easily be measured as rolling circle DNA
replication on circular templates such as the 2.9-kb Bluescript SKII
template we used in this study. The products of rolling circle
replication were separated by electrophoresis on a denaturing agarose
gel, and rates were calculated from the size distribution of the
displaced strand (Fig. 5). Surprisingly,
we noted that the difference between wild-type and exodeficient Pol Exonuclease-deficient Pol Gap Filling with Exonuclease-deficient Pol
We used a RNA-DNA primer as a model substrate for Okazaki fragment
maturation. As DNA ligase I does not ligate DNA to RNA ends,
degradation of the RNA portion is a prerequisite. Fully ligated
products were observed with almost 100% efficiency with both forms of
Pol
As noted previously, the maturation of substrates with long 5'-flaps to
which RPA can bind requires the action of Dna2 prior to FEN1, because
FEN1 is unable to slide onto flaps to which protein is bound (1-3).
The primer with the 30-nucleotide 5'-flap did not permit efficient
formation of covalently closed circles with either wild type or
exo Overexpression of Dna2 Can Rescue the Lethality in pol3-5DV
rad27-p rad51--
Based on the evidence above, Dna2 could assist in
the removal of large flaps in the cell that otherwise might be expected to lead to double strand breaks (DSBs). DSBs have been proposed to form
in the double mutant pol3-5DV rad27-p because the viability of this mutant depends on an intact DSB repair system (i.e.
a pol3-5DV rad27-p rad51 triple mutant is inviable) and can
be maintained only in the presence of a plasmid carrying wild-type
RAD27 (pRAD27) (5). To assess whether increased
levels of Dna2 would rescue the viability of the triple mutant, we
overexpressed DNA2 from a galactose-inducible promoter and
monitored loss rates of the complementing pRAD27 plasmid
(monitored by the loss of the URA3 marker as described under
"Experimental Procedures"). In agreement with a previous study (5),
cultures carrying empty vector pGAL18 showed a low frequency
of loss of plasmid pRAD27 (average = 0.4%; range
0-2% over four independent cultures). In contrast, the average loss
of pRAD27 from the triple mutant also containing
pGAL-DNA2 plasmid was 39% (range 10-61% over seven
independent cultures) but only if cells were grown on galactose to
induce overexpression of DNA2. Colonies that had lost
pRAD27 on galactose media were replated on either inducing
(galactose) or repressing (glucose) medium (Fig.
8) Thus, the viability of the triple
mutant depends on the overexpression of DNA2 because
pol3-5DV rad27-p rad51 (pGAL-DNA2) strains were able to grow only on galactose where DNA2 is
overexpressed.
The 3'-5'-Exonuclease of Pol
In the accompanying paper (3), we have described an assay to determine
the nick translation patch length, i.e. how far nick
translation proceeds past the RNA-DNA junction prior to ligation. A set
of RNA-DNA primers was used, each with a radioactive label incorporated
at a different position at 6, 12, or 22 nucleotides past the RNA-DNA
junction. A loss of label in the covalently closed product indicates
that nick translation proceeded past the labeled position prior to
ligation. Under standard replication conditions with the relevant
enzymes (Pol It is a common view that the maturation of Okazaki fragments
results from the interaction among many proteins in the cell. In this
study, we investigated the cooperation among four biochemical activities presumably involved in Okazaki maturation in
vivo. This allowed us to establish the major reaction pathways
leading to the creation of a ligatable nick. Okazaki fragments that are not ligated could lead to DSBs. Considering the large number of Okazaki
fragments (~100,000/yeast genome), even a small percentage (<0.1%)
of ligation failures might lead to a number of DSBs that would exceed
the capacity of the DSB repair system (~30 DSBs/yeast cell) and
therefore cause lethality (21, 22). An even higher reliability of
maturation is required for larger genomes such as in humans where
Okazaki fragments are expected to be 100-1000-fold more numerous and
where the number of DSBs tolerated is similar.
Although FEN1 has been established as a key activity for Okazaki
fragment maturation, additional functions can contribute to the highly
efficient maturation required for successful genome duplication. The
schematic diagram presented in Fig. 9
describes how the maturation can be accomplished, the role of FEN1 and
Dna2 in processing various intermediates, and the impact of mutants. The following discussion summarizes the in vitro and
in vivo observations that support the roles of the various
components in maturation.
and
FEN1 (Rad27) in Okazaki fragment maturation,
exonuclease-deficient polymerase Pol
-01 and Pol
-5DV
(corresponding to alleles pol3-01-(D321A, E323A)
and pol3-5DV-(D520V), respectively) were purified and
characterized in this process. In the presence of the replication
clamp PCNA, both wild-type and exo
Pol
carried out
strand displacement synthesis with similar rates; however, initiation
of strand displacement synthesis was much more efficient with Pol
-exo
. When Pol
-exo
encountered a
downstream primer, it paused with 3-5 nucleotides of the primer
displaced, whereas the wild type carried out precise gap filling.
Consequently, in the absence of FEN1, Pol
exonuclease activity was
essential for closure of simple gaps by DNA ligase. Compared with wild
type, Okazaki fragment maturation with Pol
-exo
proceeded with an increased duration of nick translation prior to
ligation. Maturation was efficient in the absence of Dna2 and required
Dna2 only when FEN1 activity was compromised. In agreement with these
results, the proposed generation of double strand breaks in
pol3-exo
rad27 mutants was suppressed
by the overexpression of DNA2. Further genetic studies
showed that pol3-exo
rad27 double
mutants were sensitive to alkylation damage consistent with an in
vivo defect in gap filling by exonuclease-deficient Pol
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Pol
)1 to carry out
DNA synthesis (1). However, biochemical experiments in the accompanying
paper (3) indicate that the main degradative force is provided
by FEN1. The activity of Dna2 becomes crucial only in cases where
strand displacement proceeds to the extent that proteins inhibitory to
FEN1 can bind to the displaced 5'-strand (2, 3).
in the process of Okazaki fragment maturation in
vivo (4, 5). This was indicated by synthetic lethality of
rad27 (FEN1) mutants with several exodeficient mutants in
Pol
and by a dramatic increase in duplication mutations in viable
pol3-exo
rad27 double mutants. We have
suggested that the 3'-5'-exonuclease could be specifically involved in
preventing the excessive formation of 5'-flaps by strand displacement synthesis.
and degradation of the displaced strand by the
nuclease activity of FEN1, a process called nick translation followed
by sealing of the nick by DNA ligase I (6). However, beside FEN1 and
Dna2, at least one more nuclease activity may function during nick
translation. It is likely that strand displacement achieved by the
5'-3' polymerization activity of Pol
is counteracted by the 3'-5'
exonuclease activity intrinsic to the polymerase. Exodeficient mutants
of T4 or T7 DNA polymerase, or Escherichia coli DNA
polymerase II carry out more efficient strand displacement than the
wild-type enzymes (7-9). In vivo, a loss of the
3'-5'-exonuclease activity of Pol
results in a large increase in
DNA duplications, which can be thought to originate from increased
5'-flap formation (5). Therefore, the 3'-5'-exonuclease activity of
Pol
may supplement the function of FEN1 and Dna2 in creating or
maintaining a ligatable nick.
beside that of replication error correction has an
important role in limiting inappropriate strand displacement synthesis
and therefore is an important determinant in creating ligatable nicks.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
01,
pol3
5DV, double mutant pol3
5DV
rad27-p, and triple mutant pol3
5DV rad27-p
rad51-null, are isogenic to CG379 (MATa ade5-1 his7-2 leu2-3,112 trp1-289 ura3-52) and have
been described previously (5). Plasmids pGAL18 (2 mM
ori URA3 GAL1-10-HA tag) and pGAL-DNA2 (pGAL18 but
GAL1-10-HA-DNA2) were used for plasmid loss
experiments (10). Pol
-5DV was purified from overproduction strain
YH712 (MATa ade5-1 his7-2
leu2-3,112::lys2D5'-LEU2
lys2::InsHS-D trp1-289 ura3-52 pol3-5DV
pep4::KanMX) carrying pBL336-5DV (2 mM ori TRP1 GAL1-pol3-5DV-(D520V))
and pBL340 (2 mM ori URA3 GAL1-POL31 GAL10-POL32) (5, 11). Because plasmid pBL336-5DV could not be
recovered in E. coli after standard subcloning procedures, the 5DV mutation was introduced into this plasmid by gap repair through
transformation of pBL336 from which a 1.4-kb
BglII-NdeI fragment surrounding the
POL3-amino acid 520 region had been removed into strain
YH712. The resulting plasmid allele was verified by PCR amplification
and sequencing of the entire plasmid POL3 gene. Pol
-01
was similarly purified from strain PY168 (Mata ura3-52 trp1-289 leu2-3,112 prb1-1122 prc1-407 pep4-3
pol3-01) carrying pBL336-01 (2 mM ori TRP1
GAL1-pol3-01-(D321A,E323A)) and pBL340 (2 mM ori URA3 GAL1-POL31 GAL10-POL32). Strain
growth, extract preparation, and purification of the mutant forms of
Pol
were as described previously for wild type (11). As a final step, the purified enzymes were passed over a Superose 6 gel filtration column to remove trace levels of a low molecular weight nuclease contamination. All other enzymes and DNA substrates were as described by Ayyagari et al. (3).
-32P]dTTP exonuclease-deficient DNA polymerase I and
Klenow fragment and purified by phenol extraction and Sephadex G50 gel filtration.
wild-type or Pol
-5DV, FEN1, Dna2, and DNA
ligase) unless indicated otherwise. In general, the DNA was
preincubated with RPA, PCNA, and RFC for 1 min at 30 °C, and the
reaction was started by adding the other proteins in a mixture.
Incubations were at 30 °C. Radiolabel was either incorporated in the
primers by extension with a single radiolabeled [
-32P]dNTP (300 Ci/mmol) as appropriate (see above) or
added as [
-32P]dATP during the replication assay. In
the latter case, the concentration of non-radioactive dATP was lowered
to 20 µM. Reaction products were analyzed by
electrophoresis on a 8% polyacrylamide 7 M urea gel on a
1% alkaline-agarose gel or a 1% agarose gel in the presence of 0.5 µg/ml ethidium bromide (12). The gels were dried and analyzed on a
PhosphorImager. Quantitation was carried out using ImageQuant
software. The images in the figures were contrast-enhanced for
visualization purposes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were initially
investigated in this study. Pol
-01 carries the classical
pol3-01 mutations (D321A,E323A) in the EXO-I motif of the
exonuclease domain, which cause a strong mutator phenotype in yeast
because of a defect in proofreading of replication errors (13, 14). In
part, this mutator phenotype can be attributed to the constitutive activation of mutagenic DNA repair in the mutant (15). The mutant is
inviable with a FEN1 deletion (rad27
) as well as with a
milder defect in FEN1 (rad27-p, F346A/F347A), which
cripples the interaction with PCNA (4, 16). On the other hand, the
pol3-5DV mutation (D520V), which localizes to the EXO-III
motif, is a less strong mutator and shows synthetic lethality with
rad27
but not with the rad27-p allele (5). The
viable pol3-5DV rad27-p double mutant has an elevated rate
of large duplications that are diagnostic of increased flap formation
rather than increased replication errors (5).
Exonuclease Reduces Efficiency of Alkylation
Damage Repair--
If the exonuclease-deficient Pol
has an
increased strand displacement capacity in vivo, it could
lead to problems during other cellular processes where flaps are
created and processed, e.g. the major pathway of base
excision repair in yeast proceeding via FEN1 and Pol
holoenzyme
(for review see Ref. 17). Therefore, we sought to determine whether the
repair of alkylation damage, which to a large extent is channeled
through the FEN1-dependent base excision repair pathway, is
impaired in the Pol
exonuclease-deficient mutant. For this purpose,
we used a viable pol3-5DV rad27-p double mutant. Whereas
neither of the single mutants is sensitive to MMS treatment, the double
mutant is exquisitely sensitive (Fig. 1).
This finding suggests that either both exonuclease activities can act
on the same DNA structure or a set of rapidly interconverting structures such as 5'- and 3'-flaps or that the exonuclease deficiency of Pol
generates aberrant DNA structures that can only be processed by a fully functional FEN1. This result also indicates that the 3'-5'-exonuclease activity of Pol
has a biological function distinct from that of proofreading of replication errors, presumably by
preventing excessive strand displacement (see below).
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Fig. 1.
Sensitivity of rad27-p pol3-5DV
double mutants to MMS. Serial dilutions of single and double
mutant strains were plated on YPD plates and on MMS-containing YPD
plates and grown at 30 °C (for details see "Experimental
Procedures").
--
Preliminary
studies with partially purified preparations had already indicated that
both mutant forms of Pol
indeed showed exonucleolytic defects (5,
13). To allow a more thorough characterization and obtain pure enzyme
useful for mechanistic studies, the mutant forms were purified to
homogeneity from yeast overexpression systems (see "Experimental
Procedures"). DNA polymerase activity was unaffected by the
exo
mutations. However, the exonuclease activity of Pol
-01 was ~0.5% wild type, whereas that of Pol
-DV was
undetectable (<0.1%) (Fig.
2A). Because Pol
-DV is
completely exonuclease-deficient, further studies were primarily
carried out with that enzyme rather than with Pol
-01, which still
shows residual 3'-5'-exonuclease activity. However, when tested in
strand displacement, nick translation, and Okazaki fragment maturation
assays described below, Pol
-01 showed activities comparable with
those of Pol
-DV (data not shown).
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Fig. 2.
Strand displacement synthesis by
exonuclease-deficient Pol . A,
DNA polymerase and 3'-5'-exonuclease activities of wild-type Pol
(wt), Pol
-01 (01), and Pol
-5DV
(5DV). B, strand displacement synthesis was
carried out under standard conditions with 20 mM NaCl and
the indicated forms of Pol
at 37 °C for the indicated times
(RPA, PCNA, and RFC were not included in this assay). Electrophoresis
was on a 8% polyacrylamide, 7 M urea gel (for details see
"Experimental Procedures").
Has Increased Strand Displacement
Activity--
For several DNA polymerases, it has been noted that
inactivation of the exonuclease activity increases the strand
displacement activity of the enzyme (7-9). These include phage T4 and
T7 DNA polymerases and E. coli DNA polymerases I and II. The
same enhancement of strand displacement was observed upon inactivation
of the exonuclease activity of Pol
. Strand displacement synthesis
by the three different forms of Pol
was carried out on a
self-priming double hairpin template, which made a rapid determination
of factors determining strand displacement synthesis possible (Fig.
2B). Under all conditions tested, both Pol
-01 and Pol
-DV were much more active in strand displacement synthesis than
wild-type Pol
. However, even strand displacement synthesis by the
exonuclease-deficient forms of Pol
was not very efficient. It
required conditions that destabilized double-stranded DNA,
i.e. elevated temperatures and low salt concentrations, and
was stimulated by elevated dNTP concentrations (data not shown).
was previously shown to depend
not only on the presence of PCNA and the clamp loader RFC but also on
the presence of the streptavidin blocks (3). In the absence of PCNA,
strand displacement synthesis by wild-type Pol
on the model
oligonucleotide substrate was very poor but detectable when the salt
concentration was at 50 mM NaCl (Fig.
3). In contrast, strand displacement
synthesis by Pol
-DV was 10-20-fold more efficient. The inclusion
of 125 mM NaCl in the assay completely inhibited strand
displacement synthesis by both DNA polymerases. However, in the
presence of PCNA, efficient strand displacement synthesis was observed
at 125 mM NaCl with both the wild-type and mutant Pol
,
although Pol
-DV was still ~4-fold more efficient.
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Fig. 3.
PCNA stimulated strand displacement
synthesis. Standard replication reactions on the
streptavidin-bound 107-nucleotide 3'- and 5'-biotinylated template
(5'-AGTGGGTTGGTTTTGGGT30CTCCCTTCTTCTCCTCCCTCTCCCTTCCCT31-3')
with a 3'-end labeled primer C12
(5'-AGGGAAGGGAGAGGGAGGAGAAGAAGGGAG-3') and a
downstream primer dc10 (5'-CCCAAAACCAACCCAC-3')
contained RPA and NaCl, PCNA and RFC, and either wild-type or 5DV
Pol as shown in the figure for the indicated times at 30 °C. The
reactions were preincubated with RPA and PCNA and RFC, if present, for
1 min at 30 °C and then started by the addition of Pol
. The
products were separated on a 7 M urea/12% polyacrylamide
gel. The length of extension products with relation to the downstream
primer is indicated above the figure.
-WT, two prominent pause sites were observed, one at the
0 position corresponding to precise gap filling and one at the +1
position corresponding to displacement of a single nucleotide by the
polymerase (Fig. 4). In sharp contrast,
Pol
-DV formed prominent pause sites at +3 to +5 positions for all downstream primers tested whether they were fully hybridized
(lane 3) or with a 5'-flap (lane 6) or whether
they were in a GC-rich region (lane 3) or in a poly(dA)
stretch (lane 8) and even when a RNA-DNA primer was used
(lane 10). This distribution of pause sites remained
constant during the course of the reaction and was similar at 30 and
22 °C (data not shown). In comparison, sequenase formed one major
pause site at the precise nick position (lane 4).
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Fig. 4.
Distinct and different pausing by
exonuclease-deficient Pol . Standard
replication reactions on the different template-primers (100 fmol each)
were as described in Fig. 3 with 125 mM NaCl. The reactions
were preincubated with RPA, PCNA, and RFC for 1 min at 30 °C and
then started by the addition of either wild type or 5DV Pol
. After
20 s at 30 °C, the reactions were stopped and analyzed by 7 M urea, 12% PAGE. The downstream primers as indicated were
dc10 (lanes 1-4), dc11 (lanes 5 and
6, 5'-C13AAAACCAACCCAC-3'), dc12 (lanes
7 and 8, 5'-A10CCCAAAACCAACCCAC-3'), and rc18
(lanes 9 and 10,
5'-p-rArCrCrCrArArArArCCAACCCAC-3'). The position corresponding to
precise gap filling is indicated with an asterisk. The
experiment in lane 4 was with 200 fmol of sequenase with no
other proteins added at 30 °C for 20 s.
is not in the rate of strand displacement synthesis but rather at an
initiation step. With Pol
-WT, replication of the primed DNA
proceeded continuously around the circle at a rate of 40-60 nucleotides/sec at 30 °C until the circle was fully replicated after
30-45 s. However, after 5 min of incubation, only 22% of the
replicated strands were longer than unit length, indicating that the
majority of replication complexes had not initiated strand displacement
synthesis. After 10 min of incubation, 50% of the complexes had
initiated strand displacement synthesis. With Pol
-DV, the same rate
of DNA replication of the SS template was observed. However, in
contrast with wild type, 63% Pol
-DV complexes had initiated strand
displacement synthesis after 5 min of incubation and 86% Pol
-DV
complexes had initiated strand displacement synthesis after 10 min. The
rate of displacement synthesis by the two enzymes was actually not
significantly different (1.5 ± 0.5 nucleotides/sec for wild type
and 2 ± 0.5 nucleotides/sec for the exodeficient enzyme).
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Fig. 5.
Pol -5DV readily
initiates strand displacement synthesis. Alkaline-agarose
electrophoretic analysis of strand displacement synthesis is shown.
Standard assays contained 100 mM NaCl. The reactions were
preincubated with RPA, PCNA, and RFC for 1 min at 30 °C and then
started by the addition of either wild type or 5DV Pol
, and
incubation was continued for the indicated times at 30 °C. (for
details see "Experimental Procedures"). The arrow
indicates fully replicated Bluescript SKII DNA (2.9 kb).
Carries Out More Rapid Nick
Translation with FEN1--
Rates of nick translation could be measured
with the same circular template primer used for strand displacement
synthesis, because the 1.14-kb-long primer is labeled at the
3'-position. Therefore, label will be lost from the DNA after nick
translation has proceeded for over 1.14-kb (Fig.
6A). An example of this
analysis is shown in Fig. 6B for Pol
-WT + FEN1. The time
point at which half of the label had been lost was taken to calculate
the average rate of nick translation. This time was corrected for the
period (~45 s) required for complete replication of the SS DNA
circle. As with strand displacement synthesis, not all complexes with Pol
-WT proceeded with nick translation. Complex
disassembly probably accounts for the ~20% label that remains even
after 20 min of incubation. Nick translation with Pol
-DV proceeded
both to a higher extent and at a higher rate. In the presence of FEN1, the rate of nick translation was 2.5 nucleotides/sec with Pol
-DV
compared with 1.7 nucleotides/sec with Pol
-WT (Fig. 6C and Table I). Those rates were not
altered by the addition of Dna2. Dna2 alone poorly supported nick
translation with Pol
-WT, and the rate with Pol
-DV was also very
low (~0.9 nucleotides/sec). Nick translation over extended stretches
of DNA as carried out in this experiment, although probably not of
physiological relevance, allowed us to determine an elongation rate for
this process because the initiation step dominates the kinetics of nick
translation through short stretches of DNA.
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Fig. 6.
Increased nick translation by Pol
-5DV and FEN1. A, schematic of the
assay. B, alkaline-agarose electrophoretic analysis of nick
translation. Standard assays contained 100 mM NaCl. The
reactions were preincubated with RPA, PCNA, and RFC for 1 min at
30 °C and then started by the addition of FEN1 together with Pol
, and incubation was continued for the indicated times at 30 °C.
The label remaining at 1.14 kb is material that did not hybridize to
the SS SKII DNA during the original priming reaction. C,
quantitation of the assay in B as well as several others.
Dna2 was added instead of FEN1 or together with FEN1 as indicated. The
same assays were also carried out with Pol
-5DV replacing wild
type.
Comparative activities of Pol -WT and Pol
-DV
Does Not Produce
Ligatable Nicks--
In the presence of PCNA, Pol
-DV appears to
produce exclusively products with 3-5-nucleotide-long 5'-flaps when
encountering a downstream double-stranded region during gap-filling
synthesis (Fig. 4). Ayyagari et al. (3) used a primed
circular SS DNA substrate to assay the accuracy of gap-filling
synthesis and Okazaki fragment maturation. DNA ligase-catalyzed
ligation of the nick resulting from precise gap filling or proper
Okazaki fragment maturation produces covalently closed circular DNA,
which has a unique migration position when electrophoresed through an
agarose gel in the presence of ethidium bromide (Fig.
7A). Indeed, when SS SKII DNA
was primed with a 5'-phosphorylated primer, replication by Pol
holoenzyme in the presence of DNA ligase I produced
predominantly (87%) covalently closed DNA (Fig. 7B,
lane 1). In sharp contrast, replication by Pol
-DV
produced no detectable covalently closed DNA (Fig. 7C,
lane 1), suggesting that the enzyme did not produce ligatable nicks. However, when FEN1 was also added to the assay, proper
processing of unannealed strand produced ligatable nicks with full
efficiency (Fig. 7C, lane 2). Dna2 was completely
inefficient (Fig. 7C, lane 3).
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Fig. 7.
Defective gap filling and Okazaki fragment
maturation by Pol -5DV. A,
schematic of the assays. B, maturation with wild-type Pol
on SS SKII DNA primed with a 5'-phosphate containing primer
(SKdc10, lanes 1-5), a RNA-DNA 5'-phosphate containing
primer (SKrc14, lanes 6-9), a 10-nucleotide 5'-flap primer
(SKdc11, lanes 10-13), or a 30-nucleotide 5'-flap primer
(SKdc12, lanes 14-17). Standard assays contained 100 mM NaCl. The reactions were preincubated with RPA, PCNA,
and RFC for 1 min at 30 °C and then started by adding the indicated
proteins in a mixture and continued for 4 min at 30 °C.
C, the same assay as in B but with Pol
-5DV
(for details see "Experimental Procedures"). Analysis was on a 1%
agarose gel with 0.5 µg/ml ethidium bromide present. Quantitation of
selected lanes is given in Table I.
when FEN1 was included in the assay, whereas Dna2 functioned
poorly with Pol
-WT and not at all with Pol
-DV (lanes
7 and 8). Maturation of a DNA primer with a
10-nucleotide 5'-flap was similar to that of the RNA primer in which
the formation of ligatable products required exclusively FEN1 in order
to degrade the 5'-flap and create a ligatable nick (Fig. 7,
B and C, lanes 11).
DNA polymerase together with FEN1 (Fig. 7,
B and C, lanes 15). More efficient
ligation was observed with the Dna2 nuclease but only for Pol
-WT
(Fig. 7B, lane 16). Pol
-DV together with Dna2 did not yield significant ligation products, and both Dna2 and FEN1
were required for efficient ligation (Fig. 7C, lanes
16 and 17).
View larger version (102K):
[in a new window]
Fig. 8.
Rescue of lethality of a pol3-5DV
rad27 rad51 mutant by overexpression of
DNA2. Strains carried the galactose-inducible
overexpression plasmid pGAL-DNA2 and either the
pRAD27-complementing plasmid (left panel) or
empty vector (right panel). Growth under inducing
(galactose) or repressing (glucose) condition was determined for serial
10-fold dilutions.
Limits the Patch Length of Nick
Translation--
The kinetics of maturation have been studied with a
model RNA-DNA-primed circular DNA substrate (see Fig. 7A)
(3). In that study (3), we noted that the maturation time,
i.e. the time required to convert nicked DNA circles into
covalently closed DNA, was substantially decreased when a large molar
excess of DNA ligase was present. In the presence of a 15-fold molar
excess of DNA ligase, the maturation time with wild-type Pol
was
15-17 s. Under the same experimental conditions, the maturation time with Pol
-DV was 18-20 s (Table I). This slightly longer maturation time could be indicative of extended nick translation by Pol
-DV prior to ligation.
, FEN1, and DNA ligase) in 50% molar excess over DNA
substrate, the measured nick translation patch length was 8-12
nucleotides for Pol
-WT and 25-40 nucleotides for Pol
-DV (a
range of three independent experiments for Pol
-WT and two for Pol
-DV). However, one major factor determining the rate of Okazaki
fragment maturation is the DNA ligase concentration. Surprisingly, a
50% excess of DNA ligase was far from saturating, and a 15-fold molar
excess of ligase was required for rapid maturation. Accordingly, with
DNA ligase in high excess, the nick translation patch was reduced to
4-6 nucleotides for Pol
-WT and 7-11 nucleotides for Pol
-DV
(Table I). Increasing the levels of the other enzymes did not
substantially alter the nick translation patch length.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 9.
Model for activities operating during
gap-filling synthesis. Cooperation among Dna2, FEN1, and the DNA
polymerase and exonuclease activities of Pol operating during
gap-filling synthesis. In this model, RPA binds to long flaps only,
thus preventing cleavage by FEN1 and stimulating cleavage by Dna2. The
crossed out enzyme shows no or poor activity on the
indicated substrate. Exo1 is shown in parentheses as it is
proposed to replace FEN1 in a rad27-
strain, but it was
not used in our studies.
Pol 3'-5'-Exonuclease Is Required for Creating a Ligatable
Nick under Conditions of FEN1 Deficiency--
This conclusion follows
from the gap-filling experiments presented in Fig. 7 and is in
agreement with the MMS sensitivity observed in a
pol3-exo
rad27 double mutant (Fig. 1)
and previous genetic studies (5). The current study established several
distinguishing properties of exonuclease-deficient Pol
that may aid
in explaining the defects of pol3-exo
mutants that
are not related to an increase in spontaneous mutation rates. A
comparison of the activities of wild type and exonuclease-deficient Pol
is given in Table I. The exonuclease-deficient Pol
is more
efficient than wild type in strand displacement synthesis, but this
increased efficiency can primarily be attributed to an initiation step
(Fig. 5). Once strand displacement synthesis has initiated, elongation
rates measured by rolling circle DNA replication are similar for both
types of enzymes. Increased strand displacement obviously would be
problematic in a cell that already is crippled for FEN1 such as in a
rad27 complete or partial mutant.
Fig. 9 represents a model of how the exonuclease activity of Pol can reduce the initiation of strand displacement synthesis. This model
is based on the assumption that when the polymerase is in the strand
displacement mode, there is a high probability of the 3'-end of the DNA
partitioning into the exonuclease site, leading to degradation of the
newly synthesized DNA back to the position of the nick. Our
observation
that pausing by Pol
-WT when a downstream
double-stranded region is encountered is at the position of the nick,
whereas pausing by Pol
-exo
is at a +3 to +4
position
is consistent with this idea (Fig. 4). This finding suggests
a model in which the wild-type polymerase is actually idling at a nick,
i.e. going though futile cycles of incorporation with
limited strand displacement followed by exonucleolytic degradation back
to the nick position. Idling by the wild-type polymerase would explain
why simple gap-filling synthesis followed by ligation is quite
successful for this enzyme, whereas it is a total failure for the
exonuclease-deficient enzyme.
Similar considerations can be put forward to explain why
maturation with just Dna2, i.e. without FEN1, gives
substantial yields of ligated products with wild-type Pol but
absolutely none with Pol
-DV (Fig. 7, compare B with
C, lanes 3, 8, 12, and
16). Dna2 specifically cuts long 5'-flaps, leaving small
5'-flaps of 5-10 nucleotides in length, and the removal of these short
flaps by Dna2 is inefficient (23). When Dna2 action is coupled to
strand displacement synthesis by Pol
, it is likely that its cutting specificity remains similar, i.e. at any given time the site
on the DNA where polymerization and degradation occur contains a small
5'-flap rather than a nick (see "Discussion") (3). Therefore, in
the absence of FEN1, a ligatable nick could only be produced through
degradation of the 3'-strand by the 3'-5'-exonuclease activity of Pol
in order to allow the displaced 5'-strand to rehybridize to the
template and produce a proper nick for ligation. The observation of a
complete failure to produce ligatable nicks by the combined action of
the exonuclease-deficient Pol
and Dna2 strongly supports this interpretation.
Cooperation between 5'-Nucleases--
In as much as
rad27- mutants are viable, an additional 5'-nuclease may
participate in creating ligatable nicks in cooperation with Pol
. A
related exonuclease, Exo1, is the most likely candidate, because
rad27 exo1 double mutants are lethal and overexpression of
EXO1 suppresses some of the rad27-
-associated
defects (24, 25). At the same time, EXO1 is unable to fully
substitute for RAD27 in the absence of the Pol
exonuclease activity, indicating a limited role for Exo1. Of course,
Dna2 is also a 5'-nuclease implicated into the maturation of Okazaki
fragments (1, 26-28). Unlike rad27-null or
exo1-null, null mutants in DNA2 are lethal and so
are mutations that inactivate the nuclease but not the helicase
activity of Dna2 (29, 30). Our biochemical results indicate that the
essential role of Dna2 is to clip long (>30 nucleotides) flaps, which
can neither be cleaved by FEN1 nor realigned by the exonuclease
activity of Pol
on the neighboring 3'-strand. In our biochemical
studies, neither wild type nor exonuclease-deficient Pol
created
detectable amounts of large flaps in the presence of FEN1. A low
frequency (<5%) of large flaps would not be detected in our
in vitro analysis, but such a low frequency of large flaps occurring in vivo, particularly in
pol3-exo
mutants, could eventually lead to the
accumulation of a lethal number of DSBs. The overexpression of
DNA2 can reduce the need for DSB repair following Okazaki
maturation and render viability to a triple mutant pol3-5DV
rad27-p rad51 (Fig. 8). Possibly, normal Dna2 levels enable
processing of only a limited number of long flaps, whereas
overexpression levels of Dna2 help Exo1 and/or the crippled FEN1-p of
the rad27-p mutant to process flaps generated by increased
strand displacement synthesis in the pol3-exo
background.
Inappropriate Strand Displacement by Exonuclease-deficient Pol during DNA Repair--
Another severe problem that may occur in
pol3-exo
strains is suggested from our observation
that gap filling by Pol
-5DV is not precise and pausing of the
mutant enzyme occurs when the enzyme enters the duplex region and
displaces a 3-5-nucleotide 5'-flap (Fig. 4). In our model system in
which simple gap maturation was measured (gap filling followed by
ligation), Pol
-5DV produced no detectable ligated products, whereas
87% ligated products were observed with Pol
-WT (Fig. 7,
lanes 1). However, in the presence of FEN1, this defect of
the mutant polymerase was completely corrected (lanes 2).
These observations may explain why pol3-5DV rad27-p mutants
are extremely sensitive to MMS damage (Fig. 1). Simple gap filling as a
final step in PCNA-dependent base excision repair requires
both FEN1 and Pol
, and in a pol3-exo
rad27-p double mutant, both degradative mechanisms that
provide a ligatable nick would be crippled (17, 31). Gap filling also occurs in the final step of nucleotide excision repair. Similarly, this
process would be expected to be defective in a
pol3-exo
strain. However, as
pol3-exo
strains are not particularly sensitive to
ultraviolet irradiation, another 5'-nuclease might function during gap
filling if Pol
is required for repair synthesis. The most probable
candidate would be RAD2, the homologue of RAD27,
which also interacts with PCNA (32). Alternatively, another DNA
polymerase such as Pol
might function generally during gap filling
or specifically in the pol3-exo
strains (33).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kim Gerik and Carrie Welch for technical assistance, Xavier Gomes for purified enzymes, and John Majors for critical discussions during the course of this work.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant GM58534 from the National Institutes of Health.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.
§ Both authors contributed equally to the work.
** To whom correspondence should be addressed. Tel.: 314-362-3872; Fax: 314-362-7183; E-mail: burgers@biochem.wustl.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M209803200
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ABBREVIATIONS |
---|
The abbreviations used are:
Pol , DNA
polymerase
;
Pol
-01, Pol
with Pol3-01 mutant subunit;
Pol
-5DV, Pol
with Pol3-5DV mutant subunit;
RFC, replication factor
C;
RPA, replication protein A;
PCNA, proliferating cell nuclear
antigen;
SS, single-stranded;
MMS, methylmethane sulfonate;
DSB, double-stranded breaks;
WT, wild type;
kb, kilobase.
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