From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Received for publication, November 26, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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Flap endonuclease 1 (FEN1), involved in the
joining of Okazaki fragments, has been proposed to restrain DNA repeat
sequence expansion, a process associated with aging and disease. Here
we analyze properties of human FEN1 having mutations at two conserved glycines (G66S and G242D) causing defects in nuclease activity. Introduction of these mutants into yeast led to sequence expansions. Reconstituting triplet repeat expansion in vitro, we
previously found that DNA ligase I promotes expansion, but FEN1
prevents the ligation that forms expanded products. Here we show that
among the intermediates that could generate sequence expansion, a
bubble is necessary for ligation to produce the expansion product.
Severe exonuclease defects in the mutant FEN1 suggested that the
inability to degrade bubbles exonucleolytically leads to expansion.
However, even wild type FEN1 exonuclease cannot compete with DNA ligase I to degrade a bubble structure before it can be ligated. Instead, we
propose that FEN1 suppresses sequence expansion by degrading flaps that
equilibrate with bubbles, thereby reducing bubble concentration. In
this way FEN1 employs endonuclease rather than exonuclease to prevent
expansions. A model is presented describing the roles of DNA structure,
DNA ligase I, and FEN1 in sequence expansion.
Repeats of simple sequences occur commonly in the human genome (1,
2). They are highly polymorphic in populations, allowing them to be
widely used as markers for gene mapping and medical purposes (3).
Within the last decade it has become more evident that these simple
repeats can lead to a characteristic type of mutation that causes human
diseases (3). Many studies have shown that the expansion of
dinucleotide repeats is associated with human cancers including those
of colon, esophagus, stomach, and lung (4-7). The expansion of
trinucleotide repeats (TNR)1
is now known to be responsible for at least 15 hereditary neurological diseases in humans (8). However, among all the possible triplet repeats, only three of them are capable of expanding:
(CAG)n·(CTG)n, (CGG)n·(CCG)n, and
(GAA)n·(TTC)n. The propensity of these sequences to
form secondary structures such as hairpins, tetraplexes, and triplexes
is thought to inhibit the activities of DNA replication and repair
proteins and lead to sequence expansion (9-11).
Several models have been proposed to explain the mechanism of TNR
expansion. Some of them are based on DNA replication (12). Others are
derived from the mechanisms of recombination, including gene conversion
and DNA repair (13). Most current information relates to the DNA
replication-based models. In the DNA slippage model, the DNA polymerase
is forced to pause by the secondary structures formed within the TNR
sequence. The delay in replication allows nascent Okazaki fragments to
transiently dissociate and reanneal in misaligned configurations,
creating hairpin and bubble structures. Elongation and ligation of
adjacent Okazaki fragments with such structures produce a sequence
expansion (14). The positive correlation among stabilities of secondary
structures, inhibition of replication proteins, and propensity for TNR
expansion supports the model (15). It is also consistent with the
finding that both CTG and CGG repeats are prone to expand when they are replicated as the lagging strand (16, 17).
The sequence expansion model proposed by Gordenin, Kunkel, and Resnick
(19) is based on the fact that inefficient cleavage of a flap by flap
endonuclease 1 (FEN1) mainly leads to DNA sequence duplication
mutations (18). In this model, DNA strand displacement synthesis
creates a TNR flap. Internal complimentarity of the repeats in the flap
allow it to form various secondary structures such as foldbacks and
hairpins. These structures inhibit FEN1 cleavage so that the enzyme
fails to resolve them. Subsequently the unresolved structures reanneal
to the template in a misaligned configuration to form a bubble
intermediate. The bubble is ligated with the upstream Okazaki fragment
by DNA ligase, generating triplet repeat expansion (19, 20).
FEN1 is a structure-specific endo/exonuclease involved in removing
initiator RNA primers on Okazaki fragments (21-24). In addition, it
has been proposed to participate in DNA long patch base excision repair
(25-27). The name of FEN1 derives from its preference for substrates
with a primer having an unannealed 5'-flap (21). The mechanism of RNA
primer removal involves creation of a flap by strand displacement
synthesis. By the current understanding of this process, part of the
flap is removed by an endonuclease named Dna2 protein. The remainder of
the flap is removed by FEN1, leaving a nick that can be ligated
(28).
The importance of FEN1 in DNA replication and repair has been
implicated by studies in vivo. In S. cerevisiae,
the FEN1 homologue is called RAD27/RTH1 (29). The
rad27/RTH1 In mice, the absence of FEN1 function results in embryonic lethality,
demonstrating that FEN1 is essential in mammals (32). Knocking out one
copy of each FEN1 and adenomatous polyposis coli genes in mice leads to
progression of adenocarcinomas. These phenotypes arise because of the
partial loss of FEN1 function in mammalian DNA replication and repair
(32).
FEN1 activity is also critical in maintaining genome stability. Genetic
studies have shown that functional defects in the 5' to 3' exonuclease
domain of Escherichia coli DNA polymerase I, a homologue of
mammalian FEN1 and Saccharomyces cerevisiae RAD27, enhances dinucleotide repeat expansion and
spontaneous mutation (33, 34). Most of these mutations are caused by
sequence duplications. The rad27 In vitro studies have been performed to further examine the
role of FEN1 in TNR expansion (20). These used various secondary structures that simulate the foldback and hairpin intermediates that
are frequently found in TNR flaps (20, 38). The results demonstrated
that both foldback and hairpin flap structures inhibit FEN1 activity.
Proliferating cell nuclear antigen (PCNA), a known stimulator of FEN1,
and replication protein A both failed to assist FEN1 to resolve these
structures (20, 39, 40). Recently, reconstituting expansion in
vitro with purified replication and repair proteins and the
substrates containing CTG repeats, we observed progressive inhibition
of CTG repeat expansion by increasing amounts of FEN1 (41). Overall
these results suggest that secondary structures in TNR flaps resist the
mechanism by which FEN1 suppresses expansions. However, in
vivo, wild type FEN1 is highly effective at inhibiting expansion.
In addition, FEN1 is involved in suppressing recombination. The
rad27 FEN1 is a highly conserved enzyme. Its homologues have been identified
from bacteriophage, bacteria, archea, yeast, and mammals including
mouse and human (44). Rad27p, the FEN1 homologue from S. cerevisiae, is highly homologous with human FEN1. Among the amino
acids of the two FEN1 homologues, 79% are conserved, and 61% are
identical (44). In addition, human FEN1 can complement most of the
phenotypes resulting from the rad27 Previously, we characterized two RAD27 mutant alleles,
rad27-G67S and rad27-G240D isolated by genetic
screening (37). These mutations are located on two highly conserved
glycines (Gly67 and Gly240) within seven
different species including bacteriophage, bacteria, yeast, mouse, and
human (44). Both mutant strains exhibit a high frequency of
dinucleotide repeat expansion and spontaneous mutation. In
vitro biochemical characterization has shown that both mutant
proteins have reduced endonucleolytic activity, have a severe
exonucleolytic defect, and failed to degrade bubbles (37). These
results lead us to propose that loss of exonuclease function of FEN1
allows sequence expansions.
Here we explore whether human FEN1 employs exonuclease to suppress
sequence expansion to further understand the roles of this enzyme in
suppressing TNR expansion directly associated with human diseases. We
took advantage of high homology and functional conservation between
Rad27 and FEN1 proteins to make point mutations in the analogous
glycines (Gly66 and Gly242) of human FEN1. We
show that the mutations cause defects that lead to sequence expansion
when the human enzyme is assayed in yeast. However, our study suggests
that FEN1 utilizes its endonuclease rather than exonuclease activity to
suppress the sequence expansion, leading us to revise our previous
hypothesis. Our results also have provided refinements to the expansion
model proposed by Gordenin, Kunkel, and Resnick (19). Based on this
model, we suggest the pathways for generating triplet repeat expansion
when FEN1 activity is compromised during DNA replication and roles of
DNA structure, FEN1, and DNA ligase I in the process.
Materials--
Yeast rad27 In Vitro Site-directed Mutagenesis of Human FEN1--
A protein
expression plasmid, FCH-pET28b, containing wild type human FEN1 open
reading frame (47) was utilized as a template for creating two point
mutations, G66S and G242D in the protein coding region. PCR-based
site-directed mutagenesis was performed using a
QuikChangeTM site-directed mutagenesis kit.
Oligonucleotides 5'-CCAGCCACCTGATGAGCATGTTCTACCGC-3' and 5'-GCGGTAGAACATGCTCATCAGGTGGCTGG-3' were used to
generate the G66S mutation, and
5'-GTATCCGGGGTATTGACCCCAAGCGGGCTGTG-3' and
5'-CACAGCCCGCTTGGGGTCAATACCCCGGATAC-3' were used to generate the G242D mutation. The substituted codons are denoted in bold
type for both primer sets, and the mutations were verified by DNA sequencing.
Construction of Plasmids and S. cerevisiae Strains and
Measurement of Dinucleotide Repeat Expansion--
To construct a
plasmid that can constitutively express wild type and mutant human FEN1
in yeast, a standard PCR method was performed to generate fragments of
ADH1 promoter and terminator using pGADT7
(Clontech, Palo Alto, CA) plasmid as a source of template sequences. Subsequently, ADH1 promoter was inserted
into SacI and XbaI sites of pRS315, and
ADH1 terminator was inserted into XhoI and
ApaI sites of the plasmid. The encoding regions of wild type
and mutant human FEN1 were amplified by PCR from plasmids FEN1-pET28b,
fen1-G66S-pET28b, and fen1-G242D-pET28b as
described above using the oligonucleotides
5'-GTCTAGAAAGCATATGGGAATTCAAGGCCTGGCC-3' and
5'-ACGCTCGAGTTATTTTCCCCTTTTAAACTTCCCTGC-3'. The
RAD27 encoding region was amplified from plasmid
RAD27-pET24b (37) by PCR using the oligonucleotides
5'-TCTAGAAAGATGGGTATTAAAGGTTTGAATGC-3',
5'-ACGCTCGAGTCATCTTCTTCCCTTTGTGACTTTATTC-3'. These
PCR primers created XbaI and XhoI sites as
underlined in the primers in the PCR products that subsequently were
inserted in between ADH1 promoter and terminator to generate
plasmids pYYL1 (RAD27-pRS315-ADH1
promoter-terminator), pYYL4 (FEN1-pRS315-ADH1 promoter-terminator), pYYL5
(fen1-G66S-pRS315-ADH1 promoter-terminator), and
pYYL6 (fen1-G242D-pRS315-ADH1
promoter-terminator). These plasmids possessing a centromere and a
LEU2 selectable marker put either wild type or mutant FEN1
under the control of the ADH1 promoter and terminator. The
plasmids were subsequently introduced into yeast EAY614 strain for
expression of mutant forms of FEN1 to measure their effects on repeat
tract instability. This created strains YL1 (RAD27), YL4
(FEN1), YL5 (fen1-G66S), and YL6
(fen1-G242D). The strains were subsequently transformed by
an indicator plasmid, pSH44, for measuring dinucleotide repeat
expansion. In pSH44, an in-frame poly(GT)16G tract is
inserted upstream of the URA3 gene (48), resulting in a
Ura+ phenotype. The poly(GT)16 repeat
expansions resulting from FEN1 functional defects can create the
frameshifts that disrupt the URA3 gene product leading to
5-FOA resistance in the strains having pSH44. The sequence expansion
rate (see Table II) was calculated from frequency of 5-FOA-resistant
colonies by utilizing the method of the median (49). The frequency data
were obtained by counting the number of surviving cells of a million
(106) either wild type or mutant cells plated on the
minimal selective medium containing 5-FOA. The experimental procedure
was the same as described before (37). Previously, we found that all of
the frameshifts in poly(GT)16G tract of pSH44 created by
rad27 Protein Expression and Purification--
Recombinant wild type
and mutant FEN1 were expressed in E. coli BL21(DE3) strain
utilizing pET-28b containing either wild type or mutant FEN1 encoding
region, which generates a C-terminal His-tagged FEN1 as described
before (37). The pellet was resuspended in lysis buffer, and bacterial
cells were lysed by three passes through a French press. The cell
lysate was subsequently sedimented for 30 min at 30,000 × g. The supernatant was loaded onto a 10-ml nickel resin
column (Qiagen, Inc.), and the proteins were purified using a fast
protein liquid chromatography system (Amersham Biosciences) according
to the procedure described by Xie et al. (37). The peak
fractions were pooled and subsequently loaded onto a Mono S column
(Amersham Biosciences) at 0.5 ml/min. The column was washed with three
volumes of HI buffer (30 mM HEPES and 0.5%
inositol) with 30 mM KCl, and the protein was eluted with a
gradient of HI buffer with 200-500 mM KCl. The peak
fractions were divided into aliquots and stored at Oligonucleotide Substrates--
The oligonucleotide primers were
designed to generate various flap and bubble substrates. The sequences
of these primers are listed in Table I.
Each substrate consists of a downstream primer (D), a template (T), and
an upstream primer (U). The 3'-end of the downstream primer of a flap
substrate is complementary to the 5'-end of its corresponding template,
whereas the 5'-end of the downstream primer is an unannealed flap. The
upstream primer of a flap substrate is complementary to the 3'-end of a
template. A flap substrate that contains a one-nucleotide flap at the
3'-end of upstream primer and also a flap at the 5'-end of downstream primer is denoted as a double-flap substrate, whereas a nick-flap substrate contains a nick in between an upstream primer and a downstream primer. For the bubble substrates the downstream primer is
annealed at both ends but with an unannealed region in the middle (see
the figure legends for detailed descriptions) to form a downstream
bubble. A nick is located between the bubble and the upstream primer. A
substrate containing CTG repeats was generated by annealing a
downstream primer containing 10 CTG repeats at its 5'-end
(DCTG10) to a template bearing 10 CAG repeats
(TCAG10). An upstream primer containing five CTG repeats at
its 3'-end (UCTG5) was annealed to the 3'-end of a
template. The substrates were constructed as described in the figure
legends.
Substrate labeling and purification were performed according to the
procedure described previously (37). Briefly, 10 pmol of a downstream
primer was 5'-radiolabeled with [ Enzymatic Assay--
The reactions with the indicated amounts of
substrates, FEN1, and/or PCNA were performed in the buffer that
contained 30 mM HEPES, 40 mM KCl, 0.01%
Nonidet P-40, 0.5% inositol, 0.1 mg/ml bovine serum albumin, and 1 mM dithiothreitol, pH 8.0. The procedure was described
previously (37). The reactions with DNA ligase I also contained 1 mM ATP as described by Henricksen et al. (41). The products were resolved on a denatured polyacrylamide gel and detected by a PhosphorImager (Molecular Dynamics). The quantitative analyses on the products were performed by using Imagequant version 1.2 software from Molecular Dynamics. The percentage of products was
calculated by the equation p = Ip/(Is + Ip) × 100, where p is the
percentage of products, Ip is the product
intensity, and Is is the starting material
intensity. All of the assays were performed in triplicate.
Enzyme Kinetics--
FEN1 kinetic experiments were performed at
30 °C under the same conditions described in the enzymatic assay.
Various amounts (5, 10, 20, and 40 fmol) of a double-flap substrate
(primers, Dflap, Tflap, and Uflap2)
and constant amounts of wild type and mutant FEN1 (2.5 fmol) were
utilized. The experiments were initiated by combining the reaction
buffer, substrates, and enzyme sequentially. 20-µl aliquots were
removed from reaction mixture at 0, 0.5, 1, 1.5, 2, and 2.5 min. The
initial velocity was determined by measuring the intensities of
substrate and product separated on a 15% polyacrylamide, 7 M urea denaturing gel using a PhosphorImager (Molecular
Dynamics). The velocity of the reaction was calculated using the same
approach as described by Tom et al. (40). The
Km and Vmax values were
obtained by fitting the data to a Michaelis-Menten equation.
Our goal has been to determine the role of FEN1 in suppression of
sequence expansion, with an emphasis on the properties of FEN1 human.
Our approach was to determine whether the human FEN1 mutants,
e.g. fen1-G66S and fen1-G242D, exhibit
a sequence expansion phenotype in vivo and display defects
in endo- and exonuclease activity in vitro. Such results
would suggest that the sequence expansion is allowed by a defect in
either endo- or exonuclease activity. Based on this, we would further
examine the substrate specificity of both wild type and mutant FEN1
proteins to distinguish the roles of FEN1 endo- and exonuclease in
suppressing the sequence expansion.
fen1-G66S and -G242D Mutants Display DNA Repeat Sequence
Expansion--
We have employed a dinucleotide repeat expansion assay
(Table II) to demonstrate that both human
fen1-G66S and fen1-G242D alleles allow
an increase in sequence expansion when expressed in S. cerevisiae compared with wild type human FEN1 and RAD27 (p < 0.01). We observed a 25- and 30-fold increase in
dinucleotide repeat expansion rates in fen1-G66S and
fen1-G242D strains, respectively, relative to
wild type (p < 0.01). The somewhat greater severity of
the phenotype of the fen1-G242D strain indicates a greater functional defect in this mutant allele. In the rad27 fen1-G66Sp and fen1-G242Dp Mutants Are Defective in Endo- and
Exonucleolytic Cleavage and Substrate Binding--
To establish the
functional defects of the human FEN1 mutant alleles, we characterized
the substrate specificity of the mutant proteins. A commonly employed
FEN1 substrate is a nick-flap substrate having a nick between a fully
annealed upstream primer and a downstream primer with an unannealed
5'-flap. Recently, a double-flap substrate having a one-nucleotide flap
at 3'-end of the upstream primer has been identified as more sensitive
to cleavage by FEN1 homologues from archeabacteria, yeast, and human
(Refs. 51 and 52 and data not shown). This substrate was proposed to be
the preferred flap structure used by FEN1 (52). We utilized a
double-flap substrate having a downstream primer with a 5'
six-nucleotide flap to measure FEN1 cleavage activity (Fig.
1). The substrate was labeled at either
the 5'- or 3'-end of the downstream primer to specifically examine FEN1
endo- or exonuclease activity, respectively. Fig. 1A
demonstrates the FEN1 endonucleolytic activity as measured by cleavage
of a 5'-end labeled substrate. Wild type FEN1 released a 7-nucleotide
product (Fig. 1A, lanes 2-8) from the
double-flap substrate corresponding to endonucleolytic cleavage at a
site 1 nucleotide into the annealed region of the downstream primer. The mutant proteins, fen1-G66Sp and fen1-G242Dp (Fig. 1A,
lanes 9-15 and 16-22), generated the same size
of endonucleolytic cleavage product as the wild type enzyme, suggesting
that substrate specificity of the enzyme had not been altered by these
mutations. However, like their yeast FEN1 counterparts, both fen1-G66Sp
and fen1-G242Dp had reduced endonucleolytic cleavage (about 20 and 10%
of substrate was cleaved, respectively) compared with the wild type
(90% of substrate was converted to products). The mutant FEN1 did not exhibit a preference for flap length because similar cleavage efficiencies were observed with nicked and 15-nucleotide nick-flap substrates (data not shown).
To supply enough enzyme for the mutant protein to exhibit
exonucleolytic cleavage, we utilized much higher levels of the wild type and mutant enzymes than those used in Fig. 1A to
titrate 3'-radiolabeled double-flap substrate (Fig. 1B).
Fig. 1B has shown that both mutant proteins exhibited severe
exonucleolytic defects as measured by cleavage of the substrate (Fig.
1B, lanes 9-14 and 16-21). Although
the endonucleolytic cleavage activities of the mutants almost reached
that of wild type at 50 fmol enzyme, they failed to generate
exonucleolytic cleavage products from the double-flap substrate
(lanes 11 and 18). This demonstrated that
effective exonucleolytic cleavage could not be achieved by simply
increasing the concentration of the mutant enzymes. Some exonucleolytic
activity was observed with G66Sp at 100, 500, and 1000 fmol. However,
these enzyme levels may not have biological significance.
To further understand how these mutations can alter enzyme function, a
kinetic study was performed (Table III).
The results showed that the fen1-G66S mutation decreased
Vmax with respect to substrate concentration by
about 5-fold and increased the Km value by about
2-fold. The fen1-G242D mutation increased
Km by about 3-fold and also decreased
Vmax by 3-fold. This indicates that the
fen1-G66S mutation mainly causes a catalytic defect but also
has a destabilizing effect on substrate binding. The
fen1-G242D mutation interfered with both substrate binding
and catalysis to a similar degree. Overall these results point out the
functional importance of the conserved glycines, Gly66 and
Gly242.
Binding assays have shown decreased substrate binding of fen1-G66Sp and
even less efficient binding for the fen1-G242Dp on the 6-nucleotide
double-flap substrate compared with wild type (data not shown). This is
consistent with the conclusions from biochemical kinetic studies
suggesting that fen1-G242Dp has a more severe substrate binding defect
than fen-G66Sp.
FEN1 Mutant Proteins Favor CTG Repeat Expansion in Vitro--
We
recently developed a system that can simulate DNA replication-related
triplet repeat expansion in vitro (41). Briefly, the
oligonucleotide substrates have overlapping upstream and downstream primers annealed to a template. The downstream primer has a
nonrepeating sequence at its 3'-end and 10 CTG repeats at its 5'-end
annealed to 10 CAG repeats on the template. The upstream primer is
annealed to the template by a nonrepeating sequence up to the
downstream primer and then is extended at the 3'-end by various numbers
of CTG repeats (CTG1 to CTG10) that can compete
with the downstream primer for annealing to 10 CAG repeats in the
template. We previously showed that the overlapping primer substrates
can form numerous structures, including bubbles that allow the ends of
the primers to form a nick, a substrate for DNA ligase I. The bubble
substrates can equilibrate into flap intermediates that can be cleaved
by FEN1, which cleaves primers at the correct length to be ligated without expansion. This ability to compete with the expansion reaction
was proposed to be a mechanism by which FEN1 prevents sequence
expansions during Okazaki fragment processing (41). To understand more
about how correctly functioning FEN1 inhibits sequence expansion, we
compared the behavior of the wild type and mutant forms of FEN1 in this
system. The downstream primers of the substrates were 3'-radiolabeled,
allowing both expanded and nonexpanded ligation products to be visualized.
Using the triplet repeat substrate where the upstream flap contains
five CTG repeats (CTG5), we examined the actions of the wild type and mutant FEN1 in the presence of DNA ligase I. A FEN1 titration was performed with the substrates using both wild type and
mutant nucleases in the presence of 2 fmol of DNA ligase I (Fig.
2). Fig. 2A shows the FEN1
cleavage and ligation products from the wild type (Fig. 2A,
lanes 4-11) and mutants (Fig. 2A, fen1-G66Sp,
lanes 13-20, and fen1-G242Dp, lanes 22-29)
resolved in a denaturing polyacrylamide gel. The amount of expanded
products was reduced as the concentrations of wild type and mutant FEN1 were increased (Fig. 2B). fen1-G66Sp or fen1-G242Dp at any
concentration with CTG5 substrate generated more expanded
products than an equivalent amount of wild type FEN1. Thus, having
reduced cleavage activity, the mutant fen1-G66Sp and fen1-G242Dp were
not as effective as the wild type FEN1 at suppressing formation of the
expansion products, indicating that a compromise in FEN1 function
favors sequence expansion. Conversely, titration of ligase I through 1, 5, 10, and 25 fmol at a constant amount of wild type FEN1 (25 fmol) on the same substrate inhibited FEN1 cleavage and led to sequence expansion (data not shown). These results are consistent with our
hypothesis that competition between FEN1 and DNA ligase I on a CTG
repeat substrate is the mechanism by which FEN1 inhibits sequence
expansion. Furthermore, they suggest that inability of the mutant forms
of FEN1 to compete with the ligation reaction accounts for the higher
rate of expansions seen in vivo.
The expanded products resulting from the mutants were not reduced to
the levels seen with wild type FEN1 (Fig. 2B). Even the highest concentrations of mutant proteins (500 fmol) used could not
deplete the expanded products to a wild type level. This was obvious
especially for fen1-G242Dp. One possibility is that a portion of the
CTG5 substrate forms relatively stable bubble structures requiring the exonuclease activity of FEN1 for resolution. In this
case, a bubble would be considered stable if the binding affinity of
its 5'-end region to the template were high enough to prevent rapid
equilibration of the bubble to a flap structure. Because the FEN1
mutants have severe exonucleolytic defects, they cannot effectively
compete with DNA ligase I to exonucleolytically remove stable bubbles.
Alternatively, FEN1 may use its endonucleolytic activity to resolve
stable bubbles by either capturing a flap transiently formed during the
equilibrium between a flap and a bubble configuration or forcing the
formation of the flap. The defective endonucleolytic activity of mutant
FEN1 may not have been robust enough to shift all the stable bubbles
into flaps and cleave them.
To explore the two possibilities, we designed a series of substrates
simulating the potential intermediates that can cause triplet repeat
expansion. On each, we examined the enzymatic activities of FEN1 and
DNA ligase I.
A Bubble Intermediate Is Essential for Generating Sequence
Expansion--
To understand the role of DNA ligase I in promoting
sequence expansion, it is critical to identify the essential
intermediates that can be directly ligated into expanded products by
DNA ligase I. These may include the hairpins and bubbles with ligatable
nicks that locate between upstream and downstream Okazaki fragments. We
tested the ligation efficiency of these structures (Fig.
3). When a substrate containing an
18-nucleotide hairpin and a nick was incubated with increasing amounts
of DNA ligase I (1, 5, 10, and 50 fmol), no expanded products were
generated (Fig. 3, lanes 7-10). This indicates that the
substrate lacks the structural requirements for ligation. Hairpin
substrates containing a gap (Fig. 3, lanes 2-5) or a
3'-flap (Fig. 3, lanes 12-15) between the upstream primer
and the downstream hairpin were utilized as controls. Neither of them
was ligated into an expanded product. A binding assay has shown that
DNA ligase I failed to bind all of these structures (data not
shown).
A bubble structure has been proposed as the intermediate that is
directly joined into an expanded product in the model by Gordenin
et al. (19). The formation of bubbles has been suggested by
studies both in vitro and in vivo (53, 54). To
simulate CTG repeat bubble intermediates, we synthesized a series of
downstream primers containing two, four, and eight CTG repeats
(CTG2, CTG4, and CTG8) that are
flanked by unique sequences at both the 5'- and 3'-ends. Each primer
was annealed to a template forcing the formation of a bubble having
CTG2, CTG4, or CTG8 repeats. The upstream and downstream primers were separated by a nick. The 3'-annealed region contained 18 nucleotides. The location of the bubbles relative to the nick between upstream and downstream primers was manipulated by increasing the length of 5'-annealed regions of the
downstream primers. In this way we generated the bubbles at a distance
of 3, 7, and 18 nucleotides from the nick to simulate annealing of
approximately one, two, and six CTG repeats at the 5'-end of CTG
bubbles. Runs of three Ts were inserted across the unannealed region of
the template.
We observed that the ligation efficiency of these substrates was
enhanced with an increase in the length of 5'-annealed region (Fig. 3,
lanes 16-30). For example, about 30% of the substrate with
the 3-nucleotide 5'-annealed region was ligated in the presence of 5 fmol of ligase I (Fig. 3, lane 18), whereas about 80% of substrates with a 7- or 18-nucleotide 5'-annealed region were converted
into ligated products by the same amount of ligase (Fig. 3, lanes
23 and 28). The improvement in ligation was almost
certainly the result of an increased stability of the bubble structure
(Fig. 3, 3-nucleotide region, lanes 17-20; 7-nucleotide
region, lanes 22-25; and 18-nucleotide region, lanes
27-30). We did not observe significant differences in
primer-template annealing stability among these substrates, suggesting
that the different ligation efficiencies are not the result of complete
dissociation of a portion of the primers (data not shown). We conclude
that formation of a bubble structure is an essential step for sequence expansion.
FEN1 Cannot Effectively Compete with DNA Ligase I on Stable CTG
Bubble Structures to Prevent Sequence Expansion--
A stable bubble
substrate with a 7- or 18-nucleotide 5'-annealed region is a poor
substrate for FEN1 endonucleolytic cleavage because it does not readily
equilibrate to a flap configuration. However, it would seem to be a
perfect substrate for FEN1 exonuclease. To learn whether human FEN1
might inhibit sequence expansion by resolving a bubble structure with
its exonuclease, we examined the exonucleolytic cleavage by wild type
FEN1 on CTG2, CTG4, and CTG8 bubble
substrates with a 7-nucleotide 5'-annealed region. We observed that 50 fmol of the wild type FEN1 was needed for sufficient exonucleolytic
cleavage to destabilize the bubble into a flap that can be removed by
FEN1 endonuclease (data not shown).
We further tested whether FEN1 can compete effectively with DNA ligase
I on a stable CTG bubble to suppress sequence expansion. CTG2, CTG4, or CTG8 bubble
substrates were incubated with increasing amounts of wild type FEN1
(10, 50, 100, and 500 fmol) and a constant, smaller amount of DNA
ligase I (5 fmol) (Fig. 4). A nick
substrate was employed as a control substrate to gauge FEN1
exonucleolytic activity. The exonucleolytic cleavage products of FEN1
are resolved as ladders (Fig. 4A, lanes 4-7).
Two types of cleavage products were generated by FEN1 from these bubble
substrates. An 18-nucleotide product results from the cleavage 1 nucleotide into the 3'-annealed region of the bubbles. The 17- and
16-nucleotide products result from FEN1-directed exonucleolytic
cleavage after the enzyme endonucleolytically removed the bubbles.
The ligation product greatly predominated over the exonuclease cleavage
product with these substrates despite the low level of DNA ligase I (5 fmol). Increasing FEN1 through 10, 50, and 100 fmol did not
significantly reduce expanded products resulting from CTG2
(Fig. 4A, lanes 11-14), CTG4 (Fig.
4A, lanes 18-21), and CTG8 (Fig.
4A, lanes 25-28) bubbles, although
dose-dependent depletion of expanded product by FEN1 was
observed. Only at the highest level (500 fmol), FEN1 removed some
annealed portion from the 5'-end of CTG bubble employing exonucleolytic
activity and significantly decreased expansion products. Notably, the
same concentration range of FEN1 had robust endo/exonucleolytic
activity on double-flap (Fig. 4A, lanes 32-35)
and nick-flap (Fig. 4A, lanes 39-42) substrates.
These results suggest that even completely functional wild type FEN1
exonuclease is ineffective at competing with DNA ligase I on the stable
bubbles. fen1-G66Sp and fen1-G242Dp essentially failed to make any cuts
in these substrates because of their severe exonucleolytic defects
(data not shown).
Because PCNA has been found to stimulate both FEN1 and DNA ligase I
(40, 55), it was possible that PCNA would help FEN1 to overcome the
dominance of DNA ligase I in these reactions. To test this hypothesis,
we titrated PCNA (0.5, 1, 2.5, and 5 pmol) on the CTG2
(Fig. 4B, lanes 17-20), CTG4 (Fig.
4B, lanes 27-30), and CTG6 (data not
shown) bubble substrates in the presence of 0.1 fmol of DNA ligase I
and 10 fmol of FEN1. We did not observe significant alteration in the
amount of expanded products generated from the substrates, even though
PCNA stimulated FEN1 exonucleolytic cleavage by a maximum of
approximately 6-fold (Fig. 4B, lanes 6 and
16) and stimulated DNA ligase I by a maximum of 5-fold (Fig. 4B, lanes 5, 15, and 25).
The same PCNA titration produces an effective stimulation on FEN1
endonucleolytic cleavage (Fig. 4B, lanes 34-37).
These results suggest that FEN1 exonuclease, although stimulated by
PCNA, cannot effectively prevent sequence expansion. Apparently, it is
FEN1 endonuclease that participates in the removal of the bubble
structures resulting from triplet repeats.
FEN1 Endonuclease Suppresses Sequence Expansion by Resolving
Unstable Bubble Structures--
To specifically examine the role of
FEN1 endonuclease in preventing CTG repeat expansion, we designed
unstable CTG bubble substrates containing two, four, or six CTG repeats
(CTG2, CTG4, and CTG6) in the
bubble region and a 3-nucleotide 5'-annealed region (Fig.
5A). Having short 5'-annealed
regions, the CTG bubbles are quite unstable, so that they can readily
equilibrate between a bubble and a flap configuration. FEN1 can use its
endonuclease activity to resolve flap structures. We have observed that
unstable CTG bubble substrates favor FEN1 endonucleolytic cleavage
(data not shown) but not ligation (Fig. 3).
DNA ligase I titration on these unstable bubble substrates at a
constant amount of FEN1 (10 fmol) was performed (Fig. 5A). With an increase in the size of the CTG bubble, the expanded products dropped from ~90 to ~10%, indicating that increasing sizes of the
unannealed region destabilizes the weakly annealed region further (Fig. 5A, lanes 4-7 versus
lanes 11-14). In addition, increasing the amount of DNA
ligase I from 5 to 50 fmol effectively inhibited FEN1 endonucleolytic
cleavage on a small CTG bubble, the CTG2 bubble (Fig.
5A, lanes 4-7). Quantitatively, 5, 10, 25, and
50 fmol of DNA ligase I inhibited FEN1 cleavage by about 30, 50, 60, and 70%, respectively (Fig. 5B), whereas the ligation products increased by a similar percentage (data not shown). Thus, DNA
ligase I can compete effectively with FEN1 to produce expanded sequences on a CTG2 bubble.
In contrast, FEN1 endonucleolytic cleavage was dominant over ligation
on unstable CTG4 and CTG6 bubble substrates
(Fig. 5, A, lanes 11-14 and 18-21,
and B). Even 50 fmol of DNA ligase only reduced the FEN1
cleavage by 5-10%. This suggests that CTG4 and CTG6 bubbles with a 3-nucleotide annealed region are so
unstable that they rapidly form flaps that succumb to the efficient
endonuclease activity of FEN1.
Both fen1-G66S and fen1-G242D mutants allow more sequence expansion
products formed from the CTG2 bubble substrate than the wild type FEN1 (Fig. 6, A,
lanes 13-20 and 22-29, and B), even when the amounts of the mutant enzyme were titrated up to 500 fmol.
This indicates that a compromise in FEN1 endonuclease activity can lead
to sequence expansion.
Unstable bubbles in a region of triplet repeats can potentially slip
further into the repeat region to form stable bubbles. These are
favorable for ligation. When FEN1 endonuclease is defective, inhibition
of sequence expansion will be compromised because the nuclease cannot
deplete unstable bubbles. Increasing the lifetime of an unstable bubble
enhances the likelihood that it will convert to a stable bubble. By
this mechanism, the endonuclease defective mutants G66Sp and G242Dp
allowed many more expanded products than wild type FEN1. Thus, we
conclude that FEN1 endonucleolytic activity suppresses triplet repeat
expansion by removing unstable bubble intermediates before they can
equilibrate into stable bubble intermediates.
Gordenin and colleagues (19) proposed a model for DNA
replication-associated sequence expansion in eukaryotes. It is based on
the currently accepted pathway for Okazaki fragment processing in which
the RNA termini are displaced into flaps by DNA synthesis from an
upstream primer. The flaps are proposed to be removed by the sequential
action of Dna2 helicase/endonuclease and FEN1 (28). In a region of
sequence such as CAG triplet repeats, the displaced flap can form
secondary structures including foldbacks and bubbles. Misaligned
annealing of the flaps on the template creates bubble and nick
structures that are ligated into expanded sequences. Subsequent repair
processes incorporate the expansion permanently into the genome.
To determine the factors that promote and inhibit triplet repeat
expansion, we developed a system in vitro that allowed
reconstitution of the expansion reactions (41). It involved two primers
that were annealed to a template and overlapped in a region of triplet repeats, simulating a displaced flap. The results showed that the
primers equilibrated into bubble structures that, upon addition of DNA
ligase I, could be joined into expanded sequences. The presence of FEN1
suppressed expansion by correctly cleaving flap intermediates to form
nicks that could be ligated at the proper length (41).
We had previously analyzed the properties of the rad27-G67S
and rad27-G240D mutant forms of the yeast FEN1homologue
(37). Cells with these mutations have frequent expansion phenotypes. The purified proteins were found to have severe defects in exonuclease function and moderate defects in endonuclease function. The loss of
exonuclease activity and the increase of sequence expansion led us to
propose that FEN1 suppresses expansion by exonucleolytic removal of the
5'- annealed nucleotides of bubble intermediates (37).
Here we have used the human FEN1 to examine the roles of FEN1
endo/exonuclease activity in suppressing sequence expansions. We also
carried out a much more comprehensive analysis of the mechanism of
repeat sequence expansions. To facilitate the analysis we generated
mutations in the human FEN1 (G66S and G242D) that are analogous to the
yeast mutants used in our previous work (37). These were shown to
support DNA replication but allow DNA sequence expansion when
introduced into a rad27 Employing the mutant human FEN1 proteins in the two-primer expansion
model system in vitro confirmed the conclusion that FEN1 suppresses expansion by competing with DNA ligase I. Although increasing concentrations of the mutant proteins decreased the amounts
of expanded products formed at a fixed level of DNA ligase, the mutants
were relatively ineffective at preventing expansion compared with the
wild type. However, this result still did not reveal the relative
importance of FEN1 endonuclease versus exonuclease activity
and the mechanism of expansion. We then specifically examined the roles
of FEN1 exo- and endonuclease, using substrates that simulate all of
the anticipated equilibrating intermediates formed by a displaced flap
in a region of repeated sequence that can directly create triplet
repeat expansion. We have done this by determining how DNA ligase I and
FEN1 compete on these substrates. A flap formed in a region having
triplet repeats can undergo slippage that allows a wide variety of
intermediates to form (54). Those that we have considered are flaps
folded back into hairpins, stable bubbles in which the 5'-annealed
region is in the 7-18-nucleotide size range, and unstable bubbles in
which the annealed region is in the 3-nucleotide size range.
Examining each in turn, we found that the folded flap with an upstream
primer cannot act as a substrate for DNA ligase, even though it
resembles the genuine ligase substrate. Previously, we also determined
that this structure inhibits FEN1 cleavage at the base of the flap (20)
and allows only a slow exonucleolytic degradation at the
double-stranded 5'-end. Therefore this intermediate is essentially
inert with respect to competition between DNA ligase and FEN1.
We made fixed structure bubble substrates that had the configuration of
the stable bubble intermediate. Because FEN1 has to initially use its
exonuclease to degrade the 5'-annealed region of the bubble, the
function of FEN1 exonuclease could be specifically tested by using
these substrates. We expected that the exonucleolytic activity of wild
type FEN1 would suppress ligation, but the mutants would be
ineffective. We did observe that a high level (at least 50 fmol) of
wild type FEN1 alone could employ exonuclease activity to slowly remove
some of the annealed portion from 5'-end of bubbles. This ultimately
destabilized the bubble into a flap that was cleaved by endonuclease
activity (data not shown). Surprisingly, however, even wild type FEN1
was a very poor competitor with ligation on these substrates. It took
500 fmol of wild type FEN1 to even moderately suppress the activity of
5 fmol of DNA ligase I (Fig. 5A). As expected, the mutants
had no effect (not shown) on the same substrates. PCNA stimulation of
FEN1 did not help to overcome the dominance of ligase in generating
sequence expansion (Fig. 5B). Overall, these results
suggested that loss of exonuclease function is not the reason why the
mutant proteins allow expansion in vivo.
In contrast, the wild type FEN1 was able to effectively cleave the
unstable bubble structure and to compete with ligation of this
substrate (Fig. 6). Differences in efficiency of endonuclease cleavage
between the wild type and mutant FEN1 are evident on this substrate,
because the mutant forms are poor endonucleases. This deficiency in
endonuclease cleavage makes the mutant species poor competitors with
DNA ligase I.
In view of these results we have revised our interpretation of the
mechanism of enzyme competition. Most likely the endonuclease function
of FEN1 allows the nuclease to capture and cleave transiently formed
flap intermediates, depleting the concentration of bubble intermediates
that can serve as ligation substrates. However, the wild type FEN1
cannot compete with ligase on very stable bubble intermediates. This is
consistent with the observation that FEN1 cannot totally suppress
expansions in the our model system in vitro, even when added
in excess and ahead of the DNA ligase I (41).
The results in vivo also show that endonucleolytic defects
of G66S and G242D allow sequence expansion. The effectiveness of partial suppression of the sequence expansion phenotype of the rad27 Our results highlight the dynamic interaction of DNA structure and
enzyme specificity in this system. Both FEN1 and DNA ligase I have
structure-specific substrate recognition. In the case of an
equilibrating substrate, the natural concentration and lifetime of a
particular structural intermediate influences the reaction rate and the
effectiveness of competition. The other important factors in a
competitive situation are the intrinsic properties of each enzyme. For
example, an unstable bubble intermediate in a CTG repeat sequence can
equilibrate with a flap configuration. FEN1 can capture the flap
intermediate or may even drive the substrate to form the flap.
Similarly the ligase may capture or stabilize the bubble. By this view,
FEN1 and DNA ligase I play active rather than passive roles in
determining triplet repeat expansion. The efficiency of each enzyme is
also important. A compromise of activity of one enzyme caused by
mutation can prevent utilization of a transient intermediate and even
shift the equilibrium in a reverse direction.
In vivo, proteins that can alter DNA structure, such as
replication protein A, a single-strand DNA-binding protein, are likely to influence the competition reaction. For example, replication protein
A should bind to the single-strand region of a large bubble and promote
dissociation of its 5'-end from the template. Recent studies suggest
that Dna2p, an endonuclease and helicase, is also involved in sequence
expansions (56, 57). Dna2p mutations in the helicase domain display
dinucleotide repeat expansion and instability of minisatellite DNA (56,
57). This suggests that the helicase activity of Dna2p destabilizes
bubbles and flap foldbacks to create a more favorable configuration for
FEN1 endo-cleavage. In addition, Dna2p has been found to physically
associate with FEN1, suggesting that the dimer of these two proteins is
more effective than FEN1 alone at promoting cleavable flap formation (58).
Our study also suggests a pathway to generate triplet repeat expansion
when FEN1 function is compromised. The endonucleolytic defect of FEN1
delays the removal of large unstable bubble intermediates. The
unresolved structures subsequently equilibrate into small stable
bubbles that can be effectively ligated into expanded sequence as shown
in Fig. 7. Because the displaced flap has
been proposed to be up to 30 nucleotides in length (59), a variety of
bubbles might form with different length of 5'-annealed and
single-stranded regions. Consistent with our interpretation, the
rad27
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant displayed phenotypes
indicative of defects in DNA replication and repair (29, 30). At a
restrictive temperature (37 °C), the growth and cell division of the
rad27
mutant stop. Morphological examination of the
mutant cells has shown that most of the cells arrest during division as
two large partially divided cells, with a blocked nuclear division,
indicating cell cycle arrest. This phenotype resembles that of
cdc2 mutants known to be defective in DNA replication. The
mutant is highly sensitive to alkylating agents, such as methyl methanesulfonate, but moderately sensitive to x-ray and UV
irradiation, suggesting that the enzyme is important in DNA base
excision repair (29, 31).
mutant also allows
frequent repeat sequence expansion. The CAG and CGG repeat sequences
associated with human neurogenetic diseases are readily expanded in the
rad27
mutant strain (35, 36). As with its bacterial
counterpart, the deletion of RAD27 increases spontaneous
forward mutation frequency by about 50-fold (18). Even partially
defective rad27 mutant strains show enhanced forward
mutation frequency (37). Interestingly, most of the forward mutations
resulting from the duplications of small direct repeats are primarily
due to the increase in repeat length (18, 37). These facts suggest that
an unresolved flap resulting from a FEN1 functional defect
reanneals to its template and forms a bubble intermediate that
generates sequence expansion.
mutant displays high recombination rates (18, 30, 42). One possible mechanism is that the unresolved flaps in rad27 mutant strains accumulate and further misalign into
loops and nicks. These flap and loop structures, if not removed by
other repair proteins, are susceptible to double-strand breaks. The presence of such breaks is known to promote recombination (18). In
fact, RAD27 has been found to be essential for viability of yeast strains having a defective double-strand break repair function (18). Furthermore, fragmented DNA was identified in rad27
mutant strains (30). The observations indicate that production of
double-strand DNA breaks is likely a mechanism that leads to increased
DNA recombination in rad27 mutants, presumably because of
the failure of resolving flap structures in these strains. Another
consideration is that the persistence of flap and loop structures
expected in rad27 mutants provides single-stranded regions
that could serve as strand invasion sites for recombination. Therefore,
one can readily envision how prompt resolution of the FEN1 flap
substrate would prevent accumulation of recombinogenic lesions. A
recent study has found that expression of human FEN1 in yeast restricts
recombination between short sequences, implying that the nuclease
participates the removal of 5' overhangs that mediate short sequence
recombination (43).
mutation in yeast demonstrating functional conservation (45). This makes the
rad27
strain a good model system to test the biological
properties of human FEN1 mutants in vivo (45).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strains, EAY614
(MATa leu
ura3-25 lys2BglII trp1
63 his3
rad27::HIS3) and EAY615 (MAT
leu
ura3-25 lys2BglII trp1
63 his3
rad27::HIS3) were obtained from Dr. Eric
Alani at Cornell University and were grown in yeast
extract-peptone-dextrose medium. The plasmid pRS315 (yeast centromere
plasmid containing LEU2 selectable marker) and pSH44
((GT)16G-URA3-ARSH4 CEN6 TRP1) were
generous gifts from Dr. Fred Sherman at University of Rochester and Dr.
Eric Alani, respectively. The strains bearing either RAD27 or human FEN1 were grown in minimal selective medium (46).
5-Fluoro-orotic acid (5-FOA) was from Zymo Research (Orange, CA) and
U.S. Biologicals (Swampscott, MA), respectively, and used as previously
described (37). Oligonucleotides were synthesized by Integrated DNA
Technologies (Coralville, IA). Radionucleotides
[
-32P]ATP (3000 Ci/mmol) and
[
-32P]dCTP (3000 or 6000 Ci/mmol) were obtained from
PerkinElmer Life Sciences. T4 polynucleotide kinase and Klenow fragment
of DNA polymerase I (labeling grade) were from Roche Diagnostics. All of the other reagents were purchased from Sigma-Aldrich and were analytical grade.
, rad27-G67S, and rad27-G240D
mutations are sequence expansions (37). Thus, the frameshift events of
poly(GT)16G occurring in FEN1 mutant strains represent the
sequence expansions created by FEN1 mutations. The genetic data shown
in Table II were statistically analyzed using the Mann-Whitney test,
where p values of <0.05 are considered significant
(50).
80 °C.
Oligonucleotide sequences
-32P]ATP and T4
polynucleotide kinase. For the 3'-end labeling of a substrate, 10 pmol
of a downstream primer was annealed to a template (50 pmol) so that a
one-nucleotide overhang was created at the 5'-end of the template. The
3'-end of the downstream primer was then extended using Klenow fragment
of DNA polymerase I and [
-32P]dCTP. The radiolabeled
primers were purified by gel isolation from either a 12% or 15%
polyacrylamide, 7 M urea denaturing gel. The substrates
were constructed by annealing a downstream primer, a template, and an
upstream primer at a molar ratio of 1:4:20, respectively.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain, the rate was increased 46-fold (p < 0.001).
Wild type human FEN1 almost completely complemented the sequence
expansion phenotype created by the rad27
mutation, with
only a 3-fold increase in sequence expansion rate compared with
RAD27. This demonstrates functional conservation between the
two FEN1 homologues. Overall, these results suggest that the mutant
FEN1 proteins are defective in removing a flap, and subsequently the
unresolved flap forms a structure that leads to sequence expansion.
Rate of dinucleotide repeat expansion in wild type and mutant FEN1
strains
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Fig. 1.
fen1-G66Sp and fen1-G242Dp have
endo/exonucleolytic defects. A, a double-flap substrate
(5 fmol) containing a six-nucleotide flap at the 5'-end of its
downstream primer and a one-nucleotide complementary flap at 3'-end of
the upstream primer was constructed by annealing primers
Dflap, Tflap, and Uflap2. The
substrate was incubated with increasing amounts (0.1, 0.2, 0.5, 0.8, 1, 2, and 5 fmol) of the wild type (lanes 2-8), fen1-G66Sp
(lanes 9-15), and fen1-G242Dp (lanes 16-22) at
37 °C for 10 min as described under "Experimental Procedures"
and separated by electrophoresis on a denaturing 15% polyacrylamide
gel. The reaction mixture in lane 1 contained only
substrate. The substrate was radiolabeled at the 5'-end of the
downstream primer. B, the double-flap substrate (5 fmol)
described in A was radiolabeled at the 3'-end of its
downstream primer and incubated with increasing amounts of human FEN1
(10, 25, 50, 100, 500, and 1000 fmol). Lanes 2-7 contained
the reactions of wild type enzyme, whereas lanes 9-14 and
16-21 contained the reactions of fen1-G66Sp and
fen1-G242Dp, respectively. Lanes 1, 8, and
15 contained only substrate. All of the reactions were
performed in a total volume of 20 µl. A schematic diagram of the
substrate is shown above the figure. The lengths of
substrate and product are shown in nucleotides, and an
asterisk denotes the position of the radiolabeled
nucleotide. nt, nucleotide.
Kinetic analysis of human FEN1
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Fig. 2.
Human FEN1 competes with DNA ligase I to
inhibit CTG repeat expansion. A, a substrate containing
five CTG repeats at the 3'-end of the upstream primer was incubated
with 2 fmol of DNA ligase I and increasing amounts of the wild type
FEN1(lanes 4-11), G66Sp (lanes 13-20), and
G242Dp (lanes 22-29) at 37 °C for 10 min. All of the
reactions were performed in a total volume of 20 µl. The substrate
was constructed by annealing primers DCTG10,
TCTG10, and UCTG5 and radiolabeled at the
3'-end of the downstream primer. The 5'-end of the downstream primer
was phosphorylated. An expanded product is denoted as the product that
is longer than its template, whereas a nonexpanded product has the same
length as its template. The bands occurring in between the
nonexpanded products and substrates are deletion products resulting
from the joining of upstream and downstream primers after the template
strand is looped out in the gap region. The gap results from
exonucleolytic cleavage by wild type FEN1. B, the data shown
in A were quantitated by Imagequant version 1.2. The
percentage of substrate converted to expanded product was plotted
against the amount of wild type and mutant FEN1. The
diamonds represent the cleavage by wild type (WT)
FEN1. The squares indicate fen1-G66Sp cleavage, whereas the
triangles correspond to fen1-G242Dp cleavage.
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Fig. 3.
Formation of a bubble structure is essential
for DNA ligase I to generate sequence expansion. The downstream
primer Dhairpin was annealed to Thairpin to
generate a hairpin substrate. Subsequent annealing of
Uhairpin1, Uhairpin2, and Uhairpin3
to Thairpin created hairpin substrates with a gap, nick, or
3'-flap in between an upstream and a downstream primer, respectively.
The 5'-end of the downstream primer contains 18-nucleotide inverted
repeat sequences that can create a hairpin structure right at the
junction where the downstream primer is annealed. Six noncomplementary
nucleotides are inserted between the inverted repeat sequences. The
substrates were 5'-radiolabeled at the downstream primers.
CTG2 bubble substrates with 3-, 7-, and 18-nucleotide
5'-annealed regions were constructed by annealing Dbubble1,
Dbubble4, Dbubble7, and Tbubble,
respectively. The upstream primers Ububble3,
Ububble2, and Ububble1 were annealed to the
3'-end of the same template to generate a nick between the upstream and
downstream primer. The 3'-annealed region of each downstream primer was
19 nucleotides long. The bubble substrates were 3'-radiolabeled and
5'-phosphorylated at the downstream primers. All of the substrates were
incubated with increasing amounts of DNA ligase I (1, 5, 10, and 50 fmol) at 37 °C for 10 min. The reaction mixtures from hairpin
substrates having a gap, nick, or flap between upstream and downstream
primers are shown in lanes 2-5, 7-10, and
12-15, respectively. The reactions from CTG bubble
substrates having 3-, 7-, and 18-nucleotide 5'-annealed region are
shown in lanes 17-20, 22-25, and
27-30, respectively. The reactions were performed in a
total volume of 20 µl. The reaction mixtures were separated on a 15%
acrylamide denaturing sequencing gel. All of the substrates are
illustrated above the figure. The asterisks
denote the positions of radiolabeled nucleotides. nt,
nucleotide.
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Fig. 4.
FEN1 exonuclease cannot effectively compete
with DNA ligase I on stable bubble structures. A,
CTG2, CTG4, and CTG6 bubble
substrates were constructed by annealing the primers
Dbubble4, Dbubble5, Dbubble6, and
Ububble2 to Tbubble. Annealing of
Dnick and Ububble1 to Tbubble
generated a nick substrate, whereas annealing of Dflap,
Uflap1, and Uflap2 to Tflap created
nick-flap and double-flap substrates. Each bubble substrate contained a
7-nucleotide 5'-annealed region and an 18-nucleotide 3'-annealed
region. The downstream primers of all the bubble substrates were
3'-radiolabeled and 5'-phosphorylated. The substrates (5 fmol) were
incubated with increasing amounts of wild type FEN1 (10, 50, 100, and
500 fmol) and a constant amount of DNA ligase I (5 fmol) at 37 °C
for 10 min. FEN1 cleavage on the nick, CTG2,
CTG4, and CTG8 bubbles are shown in lanes
4-7, 11-14, 18-21, and 25-28,
respectively. Lanes 32-35 correspond to the cleavage by
increasing amounts of wild type FEN1 (10, 50, 100, and 500 fmol) on a
double-flap substrate, whereas lanes 39-42 demonstrate FEN1
cleavage on a nick-flap substrate by the same amounts of enzyme (10, 50, 100, and 500 fmol). The reactions in lanes 1,
8, 15, 22, 29, and
36 contained only substrates. The reactions in lanes
2, 9, 16, 23, 30, and
37 contained only DNA ligase I (5 fmol) and substrates (5 fmol), whereas those in lanes 3, 10,
17, and 24 contained only FEN1 (50 fmol) and
substrates (5 fmol). Those in lanes 31 and 38 contained DNA ligase I (5 fmol) and FEN1 (50 fmol). MW
denotes a 19-nucleotide molecular marker. B, the PCNA
titration experiment was performed at a constant amount of FEN1 (10 fmol) and DNA ligase I (0.1 fmol). The ligation and FEN1 cleavage
products generated from 5 fmol of nick, CTG2 bubble, and
CTG4 bubble substrates are shown in lanes 7-10,
17-20, and 27-30, respectively. The reactions
in control lanes 2, 12, and 22 contain
5 pmol of PCNA only. Lanes 3, 13, and
23 represent the reactions containing 0.1 fmol of ligase.
Lanes 4, 14, and 24 represent
reactions only with FEN1 (10 fmol). Lanes 5, 15,
and 25 indicate the reaction mixtures containing PCNA (5 pmol) and ligase (0.1 fmol). Lanes 6, 16, and
26 represent the reaction mixtures with PCNA (5 pmol) and
FEN1 (10 fmol). PCNA stimulation on FEN1 was performed using the same
amount of PCNA (0.5, 1, 2.5, and 5 pmol), 1 fmol of FEN1, and 5 fmol of
nick-flap substrate (lanes 34-37) as described for
A. All of the reactions were performed in a total volume of
20 µl. The substrates are schematically diagramed at the
top of the figure. The reaction mixtures were separated by
electrophoresis on an 18% denaturing polyacrylamide gel. The
asterisks denote the positions of radiolabeled nucleotides.
nt, nucleotide.
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Fig. 5.
Competition between DNA ligase I and FEN1 on
unstable CTG bubble structures. A, the unstable CTG
bubble substrates were made by annealing downstream primers
Dbubble1, Dbubble2, and Dbubble3 to
the template, Tbubble. Subsequently, the upstream primer
Ububble3 was annealed to the template. Each bubble
substrate contained a 3-nucleotide 5'-annealed region and a
19-nucleotide 3'-annealed region. Increasing amounts of DNA ligase I
(5, 10, 25, and 50 fmol) and a constant amount of wild type FEN1 (10 fmol) were incubated with 5 fmol of CTG2, CTG4,
and CTG6 bubbles at 37 °C for 10 min. The downstream
primers of substrates were 3'-radiolabeled and 5'-phosphorylated.
Lanes 4-7 represent the reaction mixtures from the
substrate containing a CTG2 bubble. The reaction mixtures
from the CTG4 bubble substrate are shown in lanes
11-14. Lanes 18-21 correspond to the reactions from
the CTG6 substrate. Lanes 1, 8, and
15 indicate the reactions with substrates only. Lanes
2, 9, and 16 represent the reactions
containing DNA ligase I (10 fmol) and substrates (5 fmol). Lanes
3, 10, and 17 correspond to the reaction
mixtures having FEN1 (10 fmol) and substrates (5 fmol). The substrates
are schematically diagramed above the figure. All of the
reactions were performed in a total volume of 20 µl. The reaction
mixtures were separated by electrophoresis on an 18% denaturing
polyacrylamide gel. The asterisks denote the positions of
radiolabeled nucleotides. nt, nucleotide. B, the
results from A were analyzed by a PhosphorImager and
quantitated by Imagequant version 1.2. The percentage of substrate
converted to expanded product was plotted against the number of CTG
repeats.
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Fig. 6.
Endonucleolytic defect of FEN1 allows triplet
repeat expansion. A, the CTG2 bubble was
made as described for Fig. 5A and incubated with increasing
amounts of wild type FEN1, fen1-G66Sp, and fen1-G242Dp (5, 10, 25, 50, 100, 200, 300, and 500 fmol) and a constant amount of DNA ligase I (5 fmol) at 37 °C for 10 min. The substrate was constructed by
annealing Dbubble1 to Tbubble. Subsequently,
Ububble3 was annealed to the same template. The downstream
primer of the substrate was 3'-radiolabeled and 5'-phosphorylated. The
reaction mixtures from wild type FEN1, fen1-G66Sp, or fen1-G242Dp are
shown in lanes 4-11, 13-20, and
22-29, respectively. Lane 1 represents substrate
only control. Lane 2 contains reaction with only DNA ligase
I (5 fmol) and substrate (5 fmol). The reactions in lanes 3,
12, and 21 contain 5 fmol of substrate and 25 fmol of either wild type enzyme, fen1-G66Sp, or fen1-G242D. All of the
reactions were performed in a total volume of 20 µl. The reaction
mixtures were separated by electrophoresis on an 18% denaturing
polyacrylamide gel. The asterisks denote the positions of radiolabeled
nucleotides. nt, nucleotide. B, the results from
A were quantitated. The percentage of substrate converted to
expanded product was plotted against the amount of wild type or mutant
FEN1. The diamonds represent the cleavage by wild type FEN1.
The squares indicate fen1-G66Sp cleavage, whereas the
triangles correspond to fen1-G242Dp cleavage.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain of S. cerevisiae. Furthermore, they were shown to have severe defects in
exonuclease and moderate defects in endonuclease function, similar to
the yeast mutant proteins. The correlation between the phenotypes and
enzymatic defects of these FEN1 mutants provided clues about the
relative roles of FEN1 exo- or endonuclease in preventing sequence expansion.
mutant by fen1-G66S and
-G242D alleles approximately matches their endonucleolytic
activity. Thus, results from both in vitro and in
vivo studies led us to conclude that FEN1 endonuclease but not
exonuclease prevents triplet repeat expansion. Negritto et
al. (43) have also shown that the endonuclease rather than exonuclease activity of both yeast and human FEN1 is responsible for
inhibiting recombination between short sequences in yeast. Here we
suggest that the mechanism by which FEN1 prevents sequence expansion is
that the endonuclease removes an unstable secondary structure before it
can equilibrate to a stable bubble intermediate. However, we cannot
completely rule out the role of FEN1 exonuclease in other biological
processes in vivo.
mutation mainly allows small expansions (60),
suggesting that bubbles having small single-stranded regions are
actually ligated.
View larger version (10K):
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Fig. 7.
Proposed model of triplet repeat expansion
involving in human FEN1, DNA ligase I, and DNA structure. During
lagging strand synthesis in DNA replication, strand displacement
creates a flap having CTG repeats. Complimentarity of the repeats in
the flap allows formation of foldback or hairpin structures that cannot
be utilized as a substrate by either FEN1 or DNA ligase I. Subsequently, DNA slippage and misalignment allow an equilibrium
between the hairpin and an unstable large bubble. DNA ligase I cannot
effectively utilize an unstable large bubble having a large unannealed
region and a short 5'-annealed region. However, within a repeated
region the unstable large bubble can readily equilibrate to a stable
small bubble with a long 5'-annealed region and a short unannealed
segment. This latter structure is a poor substrate for FEN1 but a good
substrate for DNA ligase. DNA ligase I can efficiently join the nick
and generate expanded products. Alternatively, the large unstable
bubble simply forms a flap structure that is a good substrate for FEN1
cleavage. FEN1 endonuclease removes the flap, and DNA polymerization
fills a gap left by FEN1 cleavage to generate a nick. The nick is
subsequently sealed by DNA ligase I to form a nonexpanded product. DNA
sequence expansion is prevented by this means. If FEN1 endonucleolytic
cleavage is defective, the unstable bubble can equilibrate into a small
stable bubble a distance away from the nick that is favorable for
ligation allowing sequence expansion. Thus, FEN1 prevents sequence
expansion by removing an unstable secondary structure (either a bubble
or foldback) before it can equilibrate into a stable bubble
intermediate.
We observed a significant amount of expanded products generated from a small unstable bubble (CTG2 bubble with a 3-nucleotide 5'-annealed region), when equal molar ratio of FEN1 and ligase were utilized (Fig. 6). With an increased amount of ligase, FEN1 cleavage was significantly inhibited (from 90 to 20%). However, the same amounts of ligase did not affect FEN1 cleavage on larger unstable bubble structures (CTG4, CTG6 with 3-nucleotide 5'-annealed regions). This suggests that larger loops destabilize bubbles that have the same length of 5'-annealed regions. FEN1 would then be more effective at removing a larger unstable over smaller stable bubble. In vivo, another repair system, mismatch repair (MMR) is also responsible for removing bubbles. One role of MMR is to remove mismatched bases resulting from misincorporation of nucleotides during DNA polymerization. A small bubble or loop structure resulting from DNA slippage is also effectively removed by the MMR system (61). A study has suggested that one of the MMR proteins, Msh3p, even participates in repair of an unpaired loop as large as 94 bp (62). Interestingly, in regions of repeated sequence, MMR can only efficiently remove loops of 8 bases or less, and functional defects in MMR do not significantly affect the repair of loops of 16 bases or more (63, 64). A genetic study by Schweitzer and Livingston (35) also suggests that MMR in yeast only prevents small changes (1-3 repeat units) in CAG tracts including small expansions and deletions but is limited to these small sizes. This indicates that, in vivo, in regions of long repeat sequences, unstable large bubbles may be produced in Okazaki fragments. These bubbles are efficiently removed by FEN1 but not MMR proteins, presumably because they readily form a flap configuration rather than a mispaired intermediate.
Preformed triplet repeat loops resulting from meiotic recombination can successfully escape DNA repair during postmeiotic replication (53), and stable secondary structures including both hairpins and bubbles can inhibit activity of repair proteins including FEN1 (20, 38) and DNA ligase I (this study). Dna2 helicase/nuclease may have a role in disrupting foldback and bubble intermediates. However, none of these replication enzymes appear to be designed to eliminate a large stable TNR bubble. Such a structure either expands very effectively or is dealt with by recombination repair mechanisms in vivo.
In summary, our results indicate that human FEN1 endonuclease rather
than exonuclease is responsible for preventing triplet repeat
expansion. Removal of secondary structures before they become ligatable
is the mechanism by which FEN1 inhibits sequence expansion. In
addition, the competition between FEN1 and DNA ligase I on various DNA
structures is an important determinant of sequence expansion. We
propose a pathway for replication-related repeat expansion in which the
formation of a bubble structure is an essential step. Compromise of
FEN1 function allows a structure favoring FEN1 cleavage to equilibrate
into a stable bubble structure that favors ligation to an expanded
sequence. The relationship among dynamic DNA structure and the
mechanisms of FEN1 and DNA ligase I determine the frequency of expansion.
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ACKNOWLEDGEMENTS |
---|
We thank all the members of the Bambara
Laboratory for helpful discussions and suggestions, especially Dr.
Samson Tom, Dr. Janaki Veeraraghavan, Hui-I Kao, Dr. Mini Balakrishnan,
and Dr. Tamara Ranalli. We appreciate Dr. Eric Alani at Cornell
University for providing us rad27 mutant strains and
pSH44 plasmid. We also thank Sayura Aoyagi, Dr. Jeffrey J. Hayes, and
Dr. Fred Sherman at the University of Rochester for the generous gifts
of recombinant human DNA ligase I and pRS315 plasmid.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM24441.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: University of
Rochester Medical Center, Dept. of Biochemistry and Biophysics, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 585-275-3269; Fax: 585-271-2683; E-mail: robert_bambara@urmc.rochester.edu.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M212061200
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ABBREVIATIONS |
---|
The abbreviations used are: TNR, trinucleotide repeat; FEN1, flap endonulease 1; PCNA, proliferating cell nuclear antigen; 5-FOA, 5-fluoro-orotic acid; MMR, mismatch repair.
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