(Received for publication, July 10, 1995)
From the
The 5` 3`-exonuclease domain of Escherichia coli DNA polymerase I is required for the completion of lagging strand
DNA synthesis, and yet this domain is not present in any of the
eukaryotic DNA polymerases. Recently, the gene encoding the functional
and evolutionary equivalent of this 5`
3`-exonuclease domain has
been identified. It is called FEN-1 in mouse and human cells
and RTH1 in Saccharomyces cerevisiae. This 42-kDa
enzyme is required for Okazaki fragment processing. Here we report that
FEN-1 physically interacts with proliferating cell nuclear antigen
(PCNA), the processivity factor for DNA polymerases
and
.
Through protein-protein interactions, PCNA focuses FEN-1 on branched
DNA substrates (flap structures) and on nicked DNA substrates, thereby
stimulating its activity 10-50-fold but only if PCNA can
functionally assemble as a toroidal trimer around the DNA. This
interaction is important in the physical orchestration of lagging
strand synthesis and may have implications for how PCNA stimulates
other members of the FEN-1 nuclease family in a broad range of DNA
metabolic transactions.
In eukaryotic cells, a family of structure-specific
endonucleases can be defined based on conserved domains within FEN-1
(flap endonuclease), a 42-kDa enzyme that is both a
5` flap DNA endonuclease and a nick specific
5`-exonuclease(1) . The Saccharomyces cerevisiae analog of FEN-1 is encoded by the RTH1 gene (2, 3) . Both human and yeast FEN-1 (yFEN-1) ()are highly homologous to the human DNA repair gene XP-G and its yeast homologue RAD2(4) . Various DNA metabolic processes are thought
to require processing of intermediates by the FEN-1 endonuclease. The
enzyme shows the greatest activity as an endonuclease on nicked
double-stranded DNA substrates with the 5`-end of the nick expanded
into a single-stranded tail (see structure in Fig. 2A),
cutting these so-called flap structures at the base of the
tail(5) . These types of DNA intermediates likely occur during
end joining reactions in which DNA ends with limited homology are
joined. Mammalian FEN-1 has also been identified as DNase IV, or
maturation factor I, a nick-specific 5`
3`-exonuclease required
for nick translation during Okazaki fragment
maturation(6, 7, 8, 9, 10) .
Consistent with its corresponding functional activity, mammalian FEN-1
shows sequence homology with the 5`
3`-exonuclease domain
present in Escherichia coli DNA polymerase I(10) . The
yeast RTH1 gene is dispensable for cell growth, but rth1 deletion mutants are temperature-sensitive for growth and show a
terminal phenotype consistent with a defect in DNA
replication(2, 3) . Genetic studies with yeast rth1 deletion mutants have also shown that FEN-1 functions in
the repair of alkylation damage and in recombinational repair. However,
yFEN-1 (RTH1) does not function in nucleotide excision
repair(2, 3) . Rather, in vitro studies have
shown that the endonucleolytic activity of XP-G (the mammalian
homologue of RAD2) is required for this repair
process(11) .
Figure 2:
PCNA
stimulates yFEN-1 endonuclease activity on a flap substrate. A, diagram of a DNA flap substrate. The position of the label
is indicated by the asterisk on the flap strand, SC5.
Oligonucleotides are: SC1, CAGCAACGCAAGCTTG (strand adjacent to the
flap strand); SC3, GTCGACCTGCAGCCCAAGCTTGCGTTGCTG (strand annealed to
the flap and the adjacent strand); and SC5,
ATGTGGAAAATCTCTAGCAGGCTGCAGGTCGAC (flap strand, which is the one
labeled) (see (1) for a full description). The 5` ends are
indicated. B, stimulation of yFEN-1 endonuclease by PCNA. The
endonuclease assay was done in a 15-µl total volume containing 50
mM Tris-HCl, pH 8.0, 10 mM MgCl, 0.5
mM
-mercaptoethanol, 500 µg/ml BSA, 10 fmol of flap
substrate, 50 fmol of FEN-1, and, when present, 26,000 fmol as trimer
of PCNA (2.3 µg), 30 fmol of RF-C, and 100 µM ATP. C, mutant PCNA fails to stimulate. The assay was done as in panelB, but the PCNA concentration was varied as
indicated. pcna-52, which is a monomer in solution, was added at the
indicated level. Products of the reaction were separated on a
denaturing 15% polyacrylamide gel and visualized on a PhosphorImager.
The mobility of the top band is 33 nucleotides and that of the cleaved
product is 20 nucleotides. Control experiments showed no detectable
nuclease activity by PCNA alone (data not
shown).
The yeast proliferating cell nuclear antigen
(PCNA) is the processivity factor for DNA polymerases and
.
It is a homotrimer with a subunit molecular mass of 29 kDa and is
highly conserved from yeast to mammalian cells. The crystal structure
of yeast PCNA shows that the trimer forms a closed ring with the
appropriate dimensions and electrostatic properties to encircle
double-stranded DNA and to interact with it using nonspecific
contacts(12) . Processivity in DNA synthesis is achieved by
protein-protein interactions between PCNA and the polymerase, thereby
tethering the DNA polymerase at the primer terminus(13) . In
addition to this structural function during the elongation phase of DNA
replication, mammalian PCNA, through its interactions with the
cyclin-dependent protein kinase inhibitor p21 (CIP1/WAF1/SDI1), has
also been implicated in cell cycle control(14, 15) .
In this communication we show that PCNA physically interacts with FEN-1
and sequesters it to its site of action, thereby stimulating the
activity of FEN-1 10-50-fold.
The RTH1 gene was isolated by us as a PCNA-interacting gene using the yeast two-hybrid method(20) . In this search for interacting genes, the bacterial lexA DNA binding domain was fused to a cold-sensitive PCNA mutant (pol30-52) rather than to the wild-type POL30 gene as strains with the latter construct grew poorly and had a very low transformation frequency(17) . The measured interaction signals between POL30-lexA or pol30-52-lexA and RTH1 fused to the GAL4 activation domain were identical and weak (see ``Experimental Procedures''). As the two-hybrid method may detect both direct and indirect interactions, we turned to biochemical methods to investigate a possible interaction between yFEN-1 and PCNA.
The existence of a specific protein-protein interaction between
yFEN-1 and PCNA was confirmed by affinity chromatography on PCNA beads.
Yeast FEN-1 bound specifically to PCNA beads but not to control BSA
beads (Fig. 1A). Elution from the PCNA beads was
accomplished at 0.6 M NaCl, indicating that salt bridges
contribute substantially to the PCNA-FEN-1 interaction. Subsequent
treatment with ionic detergents did not release additional yFEN-1 (Fig. 1A). The specificity of the interaction was
demonstrated by the observation that an excess of PCNA blocked binding
of yFEN-1 to the beads (Fig. 1B). As expected from the
observed in vivo interaction between yFEN-1 and the mutant
pcna-52 by the two-hybrid method, an excess of the latter mutant
protein also blocked the binding of yFEN-1 to PCNA beads. Finally, we
showed that the beads can bind yFEN-1 from crude yeast extracts. That
the species detected on the Western blot is yFEN-1 follows from its
comigration with purified yFEN-1 and the absence of a signal with rth1 extracts (Fig. 1C). Although yFEN-1
is not the major polypeptide species from crude extracts bound to PCNA
beads, it can be detected as a distinct band on silver stained gels,
which is absent when extracts are used from an isogenic
rth1 strain (data not shown).
Figure 1: Immunoblot analysis of yFEN-1 binding to PCNA beads. A, fractionation of yFEN-1 on BSA or PCNA beads. See ``Experimental Procedures'' for details. B, competition assay. The assay was as described, except that yFEN-1 was preincubated for 10 min at 4 °C with 100 µg of BSA (lane1) or 30 or 100 µg of PCNA (lanes2 and 4) or 30 or 100 µg of pcna-52 (lanes3 and 5) in 100 µl of buffer A prior to addition of PCNA beads. The 0.6 M NaCl eluate was analyzed. C, PCNA beads bind yFEN-1 in crude extracts. Extracts (500 µg) were incubated with 10 µl of PCNA beads in 200 µl of buffer A, washed, and eluted with a total of 20 µl of buffer A containing 0.6 M NaCl as described. Lane1, 15 ng of purified FEN-1; lane2, 10 ng of FEN-1 plus 10 µl of 0.6 M NaCl eluate from strain PY26; lane3, 10 µl of eluate from strain PY26; lane4, 10 µl of eluate from strain PY59.
The functional interaction between yFEN-1 and PCNA was probed with DNA substrates that are probable intermediates in DNA end joining (flap structures, Fig. 2A) and in DNA replication (nicked duplexes). On a model flap structure, PCNA stimulated the activity of yFEN-1 about 10-fold (Fig. 2B) based on PhosphorImager quantitation. It is important to note that, in contrast to assays with nicked substrates described below, only PCNA and yFEN-1 were added in this assay. Therefore, stimulation of yFEN-1 activity can be directly attributed to its interaction with PCNA rather than with other accessory factors. Stimulation requires that PCNA is a trimer. The pcna-52 mutant exists as a monomer in solution(17) . Despite the fact that this mutant protein exhibits a similar affinity for yFEN-1 as wild-type PCNA, it failed to stimulate yFEN-1 activity (Fig. 2C; see also Fig. 1B). The most straightforward conclusion of these experiments is that PCNA must encircle the double-stranded DNA in order to stimulate yFEN-1, and the mutant form is unable to do so. Because this mode of PCNA loading occurs by nonspecific diffusion onto DNA ends, a large excess is required to observe substantial stimulation(21) . Thus, at the lowest concentration of PCNA tested (0.02 µg), which represents a 20-fold molar excess over DNA substrate, very little stimulation was observed (Fig. 2C). RF-C is required for the efficient loading of PCNA at primer termini in an ATP-dependent manner(18, 22, 23, 24) . If RF-C would efficiently and appropriately load PCNA at flap structures one would expect to observe stimulation of yFEN-1 activity at PCNA levels stoichiometric with DNA substrate. However, inclusion of RF-C and ATP in the nuclease assay did not give a significant further stimulation of yFEN-1 beyond that observed by PCNA alone, either at high concentrations of PCNA (Fig. 2B) or at low concentrations of PCNA, which show only minimal stimulation (data not shown). Possibly, RF-C fails to recognize the flap structure as a docking site for PCNA. Or, alternatively, PCNA loaded by RF-C is on the wrong double-stranded side of the flap structure necessary for yFEN-1 stimulation. These observations with the yeast enzymes were extended to the human system. Human PCNA stimulated the endonucleolytic activity of human FEN-1 on the flap structure substrate to a similar degree (data not shown).
In contrast to these results with flap substrates, we
observed no obvious stimulation of yFEN-1 activity by PCNA on model
oligonucleotides containing a nick (data not shown). ()PCNA
is known to slide rapidly across linear DNA molecules(25) . As
the more rapid sliding of PCNA across these small linear
double-stranded DNA substrates, in comparison to the sterically
hindered flap substrates, might not provide a significant mean
residence time for PCNA in order to interact productively with yFEN-1,
we turned to circular DNA substrates from which PCNA, once loaded,
would not be able to dissociate (Fig. 3). RF-C and ATP are
absolutely required to load PCNA at primer termini of circular
substrates(21) . In agreement with these known properties of
PCNA, we observed stimulation of yFEN-1 activity by PCNA but only if
RF-C and ATP were also present. Interestingly, the inclusion of salt
(75 mM NaCl) in the assay revealed the functional interaction
between PCNA and yFEN-1 most profoundly. As PCNA is efficiently loaded
by RF-C onto the DNA at physiological salt levels, it in turn is
capable of loading yFEN-1 and hence stimulating its activity (Fig. 3). In the absence of PCNA, yFEN-1 fails to interact with
the DNA at these salt concentrations, and a modification of the assay
to very low salt and magnesium concentrations is essential to detect
yFEN-1 activity at nicks(5) . No stimulation was observed with
the monomeric mutant pcna-52 (data not shown).
Figure 3:
Stimulation of FEN-1 by PCNA at DNA nicks.
A 5`-labeled 24-mer oligonucleotide and a 36-mer oligonucleotide,
representing positions 6353-6330 and 6329-6294,
respectively, were hybridized to single-stranded mp18 DNA (upperstructure). Assays were as described in the legend to Fig. 2, except for adjustment to 10 mM Tris, pH 8, 5
mM MgCl, and 75 mM NaCl. In a 15-µl
total volume, 20 fmol of substrate coated with 400 ng of E. coli single-stranded DNA binding protein and, where indicated, 100 fmol
of FEN-1, 1300 fmol of PCNA, 30 fmol of RF-C, and 100 µM ATP were incubated for 10 min at 30 °C. Analysis was as
described in the legend to Fig. 2. The topband is the labeled 24-nucleotide oligonucleotide. The bottomband (arrow) is the released 5` mononucleotide.
Control experiments showed no nuclease activity in all analogous
experiments lacking FEN-1 (data not shown).
The interaction
between FEN-1 and PCNA may have bearing on the structure of the lagging
strand DNA replication complex. Because FEN-1 does not negatively
affect DNA synthesis by DNA polymerase or
holoenzyme (i.e. does not compete with these polymerases for binding to
PCNA), (
)it may form an integral part of this complex and
mediate coupled synthesis and maturation of Okazaki fragments (Fig. 4). In the presence of DNA polymerase
or
, the
complex would carry out nick translation until DNA ligase I seals the
nick(9) . In mammalian cells, an RNase H is also required for in vitro Okazaki fragment maturation(7, 26) .
However, it is not known at this time whether this enzyme forms an
integral component of this maturation complex.
Figure 4:
Hypothetical positioning of PCNA and
FEN-1 at recombinational flap intermediates (left) and during
Okazaki fragment maturation (right). RF-C and polymerase
are also shown in the Okazaki fragment model. Indicated contacts with
PCNA are based on physical and functional interaction
studies.
In addition to replication, the interaction between FEN-1 and PCNA may have broader implications in DNA metabolism as well. In nucleotide excision repair, for example, PCNA has been shown to play an important role(27, 28) . Although FEN-1 likely is not involved in this reaction, a highly homologous structure-specific nuclease, XP-G or RAD2, is absolutely required. Based on the involvement of PCNA in nucleotide excision repair and the presence of homology between FEN-1 and RAD2, it is possible that PCNA may also interact with RAD2 to facilitate its loading and thereby excision of damaged nucleotides. Thus, there may be a common theme in various aspects of DNA metabolism, in addition to DNA replication, in which a processivity factor stimulates a structure-specific nuclease in processing nicked and branched DNA intermediates.
Based on the studies here, we conclude that the functional interaction between PCNA and FEN-1 is important in the orchestration of lagging strand processing at the eukaryotic DNA replication fork. The interaction of PCNA with other FEN-1 family members may be generally important to a wide variety of transactions involving branched DNA intermediates.