(Received for publication, January 8, 1997, and in revised form, February 18, 1997)
From the Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435
Using a synthetic telomere DNA template and whole
cell extracts, we have identified proteins capable of synthesizing the
telomere complementary strand. Synthesis of the complementary strand
required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy- and ribonucleosidetriphosphates and was
inhibited by neutralizing antibodies to DNA polymerase . No evidence
for RNA-independent synthesis of the lagging strand was observed,
suggesting that a stable DNA secondary structure capable of priming the
lagging strand is unlikely. Purified DNA polymerase
/primase was
capable of catalyzing synthesis of the lagging strand with the same
requirements as those observed in crude cell extracts. A ladder of
products was observed with an interval of six bases, suggesting a
unique RNA priming site and site-specific pausing or dissociation of
polymerase
on the d(TTAGGG)10 template. Removal
of the RNA primers was observed upon the addition of purified RNase HI.
By varying the input rNTP, the RNA priming site was determined to be
opposite the 3
thymidine nucleotide generating a five-base RNA primer
with the sequence 5
-AACCC. The addition of UTP did not increase the
efficiency of priming and extension, suggesting that the five-base RNA
primer is sufficient for extension with dNTPs by DNA polymerase
.
This represents the first experimental evidence for RNA priming and DNA
extension as the mechanism of mammalian telomeric lagging strand
replication.
Semiconservative discontinuous bidirectional DNA replication
presents the "end problem" for replication of linear genomes (1).
Mammalian chromosomes contain short repeated DNA sequences complexed
with specific proteins at each termini (telomeres) and, in conjunction
with telomerase, can circumvent this problem. Telomerase has been
extensively studied and catalyzes the 5 to 3
extension of the
terminal 3
-OH using an internal ribonucleotide template (reviewed in
Ref. 2). To completely replicate telomeres, the strand complementary to
the telomeric leading strand must be synthesized to generate a double
strand DNA product.
The mechanism by which the DNA strand complementary to the telomerase
catalyzed leading strand is synthesized has not been addressed
experimentally. Two models have been proposed for synthesis of the
telomere lagging strand. The first involves RNA priming and DNA
extension followed by removal of RNA primers and ligation of the
Okazaki-like fragments (3, 4), presumably using the same enzymatic
machinery employed for lagging strand synthesis at a replication fork
(5, 6). The alternative hypothesis is based upon in vitro
analyses of telomeric DNA sequences and the propensity to form higher
ordered structures (7). The structure generated must present a
3-hydroxyl for DNA polymerase (pol)1
catalyzed extension for synthesis of the lagging strand followed by a
nucleolytic processing reaction to generate a terminal 3
-hydroxyl to
enable telomerase extension in the next round of replication. Experimental evidence for either of these hypotheses or other alternative mechanisms has not been obtained. The goal of this study
was to identify proteins in mammalian whole cell extracts and determine
the mechanism for copying a synthetic mammalian telomeric
substrate.
Unlabeled nucleotides were from Pharmacia Biotech Inc., and
radiolabeled nucleotides were from DuPont NEN. HeLa whole cell extracts
were prepared essentially according to Wood et al. (8). Calf
thymus DNA pol /primase was purified by immunoaffinity
chromatography according to Nasheuer and Grosse (9). Calf thymus RNase
HI was generously provided by R. Bambara (University of Rochester). Calf DNA ligase I and FEN-1 were purified as described previously (10).
The TS10, consisting of 10 repeats of the sequence d(TTAGGG) was
synthesized on a Molecular Biosystems 390 DNA synthesizer and purified
by 15% polyacrylamide/7 M urea preparative DNA sequencing gel electrophoresis (11). Lagging strand synthesis reactions were
performed in 10 mM Tris-Cl, pH 7.5, 5 mM
MgCl2, 7.5 mM dithiothreitol, 1 mM
each dATP, dGTP, and dTTP, and [
-32P]dCTP (1 µCi, 10 µM). Reactions were supplemented with 5 pmol of TS10
telomere substrate, 1 mM rNTPs, and protein as indicated in
the figure legends. Reactions were incubated for 60 min at 37 °C and
terminated by the addition of EDTA to 0.1 M, NaOAc to 0.3 M, 1 µg of glycogen, and 100 µl of absolute ethanol.
Reaction products were collected by sedimentation at 12,000 × g for 20 min at 4 °C and dissolved in 10 µl of 50%
formamide, 0.1 M EDTA, 0.01% each of bromphenol blue, and
xylene cyanole. Products were separated by electrophoresis through 15%
polyacrylamide/7 M urea DNA sequencing gels. Gels were
dried, and products were visualized by autoradiography.
We have initiated the study of how mammalian cells complete
replication of linear chromosomes using an in vitro
approach. A synthetic single-stranded DNA substrate, TS10, was
constructed consisting of 10 repeats of the human telomeric DNA
sequence d(TTAGGG). The TS10 DNA substrate was incubated with crude
cell extracts prepared from exponentially growing HeLa cells. HeLa
cells have been demonstrated to contain active telomerase and have
stable telomere lengths (12). Therefore, these cells were used to
identify proteins that are capable of copying the telomere leading
strand template. Synthesis of the complementary strand requires only dATP, dTTP, and dCTP; therefore we employed conditions to measure the
incorporation of [-32P]dCTP into DNA, dependent upon
the input DNA template. Whole cell extracts often contain some
endogenous DNA that can serve as a template or primer for DNA
synthesis. Therefore, reactions were performed in the presence and the
absence of added TS10 substrate to identify products generated that are
solely dependent on the input DNA. The results shown in Fig.
1 demonstrate that in the absence of added TS10
substrate, a product of approximately 22 bases is observed (lane
1). In the presence of the telomere DNA but without the addition
of rNTPs, the 22-base product is also observed with no other products
identified (lane 2). The product migrating at 22 bases was
not susceptible to degradation by DNase or RNase. However, the majority
of the product could be removed by protease degradation and phenol
extraction prior to electrophoresis. Incorporation of the dCTP label
into products other than the 22-base telomere-independent product was
observed in reactions containing rNTPs and the TS10 substrate
(lane 3). Products were observed at one-base intervals
ranging from 5 to 30 nucleotides in length in addition to minor
products at 35, 42, 48, and 55 bases. The dependence on rNTPs strongly
suggests that an RNA priming event is required for DNA extension. No
products were observed greater than 60 bases in length, indicating that
the TS10 substrate itself was not being extended via formation of a
hairpin secondary structure. To identify the proteins responsible for
the synthesis of the telomeric lagging strand, we employed the
monoclonal antibody SJK 132-20, which inhibits DNA pol
and shows no
cross-reactivity with DNA pols
and
(13). Preincubation of the
cell extract with the inhibitory antibody abrogated the telomere
DNA-dependent DNA synthetic activity (lane 4),
demonstrating that DNA pol
is responsible for generating the
telomeric lagging strand products.
Because the synthetic activity associated with the telomeric template
was dependent on DNA pol and rNTPs, an RNA priming mechanism was
likely. DNA pol
has a tightly associated primase activity capable
of de novo RNA priming (reviewed in Ref. 14). Therefore, we
purified DNA pol
/primase from calf thymus by immunoaffinity chromatography (9). The purified DNA pol
/primase was then assayed
for the ability to perform lagging strand synthesis on the TS10
substrate. The results shown in Fig. 2A
demonstrate that in a complete reaction containing TS10, rNTPs, and
dNTPs, increasing concentrations of calf DNA pol
/primase were able
to catalyze the synthesis of DNA using the telomeric DNA as a template.
Quantification of these results demonstrate a linear increase in
incorporation. The results presented in Fig. 2B demonstrate
that DNA pol
/primase requires rNTPs and TS10 to generate DNA
synthetic products (lane 1), because reactions performed
without rNTPs (lane 2) or without TS10 (lane 3)
show no synthesis. The products observed from DNA in the reaction with
purified DNA pol
/primase synthesis occur in groups of three bases.
These products are likely the first, second, and third dCMP label being
incorporated. The low concentration of dCTP employed is necessary to
achieve the specific activity required to efficiently detect synthesis
and likely results in dissociation or pausing of DNA pol
/primase as
a result of low dNTP concentration and an inherent low processivity of
DNA pol
(15). The products are also observed at six-base intervals centered approximately at 16, 22, 28, and 34 bases, a similar length
distribution to that observed in the cell extract (Fig. 1). This
pattern suggests a unique primer initiation site and DNA synthesis
termination site. The site-specific termination or pausing is likely in
the run of three guanosine bases in the template strand, as discussed
earlier. In addition, DNA synthetic products generated by calf DNA pol
/primase on the TS10 substrate could be extended further by the
addition of the large fragment of Escherichia coli DNA pol I
(data not shown). This result provides evidence that termination or
pausing observed is not the result of complete synthesis over the
entire template. The difference in small products observed in the cell
extract compared with the purified DNA pol
is likely the result of
contaminating nucleases present in the extract.
Removal of RNA primers from lagging strand Okazaki fragments is
necessary for complete processing and ligation. The eukaryotic pathway
for removal of RNA primers requires both RNase HI and FEN-1 (5, 6).
Therefore, we employed purified calf RNase HI in complete lagging
strand synthetic reactions. The results shown in Fig. 3
demonstrate an increased mobility of products (lanes 2-4)
compared with reactions performed without RNase HI (lane 1).
The decrease in intensity of products observed at 26, 32, and 38 bases
is accompanied by an increase in products at intermediate positions and
low molecular weight. These results demonstrate that the RNA primers
are at least partially processed by RNase HI. Complete processing
requires FEN-1 and DNA ligase I in addition to RNase HI (5, 6).
Reactions were performed with each of these components, and the results
are also shown in Fig. 3. The addition of RNase HI and FEN-1 results in
a further increase in mobility (lane 6) compared with
reactions performed without RNase H. These results are consistent with
the role of FEN-1 in removing the last ribonucleotide from an Okazaki
fragment (6, 10). The addition of DNA ligase I to the reactions had essentially no effect (lane 7). The inability to observe
ligation products likely results from not having multiple priming
events on a single template. Each reaction contains 5 pmol of TS10
substrate, and we estimate that less than 5% of the substrates are
utilized during the course of the reaction, thereby decreasing the
likelihood of a single DNA molecule sustaining two priming events.
Although RNase HI can degrade RNA primers from RNA-DNA hybrids, RNase H from Drosophila melanogaster alters its cognate DNA pol
/primase activity increasing primer synthesis (16). A similar
interaction has also been observed with RNase H and DNA pol
from
calf (17). Interestingly, using calf thymus RNase HI purified by a
different method (18), we have observed the stimulation of DNA pol
/primase (data not shown). The relationship between these two forms
of RNase H and their interaction with DNA pol
is currently under investigation.
The specific banding pattern observed in telomere lagging strand
reactions suggests that primer initiation may be at a unique site on
the telomeric DNA template. Previous studies have shown that DNA pol
/primase initiates primer synthesis by creating a dinucleotide
complex with a single-stranded template (19). DNA pol
/primase acts
by first adding the eventual second nucleotide of the RNA sequence,
preferring this nucleotide to be a purine. More recently, a
preferential priming sequence 5
-d(GCTTTCTTCC) has been deduced
in vitro (20). In vivo experiments mapping replication initiation sites identified a similar sequence that serves
as a preferential priming site (21). The telomeric d(TTAGGG) repeat
contains only two pyrimidines, and if sequence-specific initiation is
occurring, it is likely to be opposite the two thymidine bases. To test
this hypothesis, we performed an experiment varying the rNTPs added to
the lagging strand reactions. The results are shown in Fig.
4 and demonstrate that DNA pol
/primase catalyzed priming and extension in reactions containing all four rNTPs
(lane 1). In addition, reactions performed with only rATP,
rCTP, and rUTP (lane 2) and rATP and rCTP (lane
5) resulted in a similar distribution of products and rate of
incorporation to that observed with the full complement of rNTPs
(lane 1). The synthetic activity observed in reactions
performed with only rATP and rCTP (lane 5) was 50% greater
than that observed in reactions performed with the full complement of
rNTPs (lane 1). Interestingly, all reactions containing rCTP
(lanes 3 and 6) supported approximately 10-20% of primer synthesis and extension compared with reactions performed with all four rNTPs. CTP also alone resulted in this low level of
priming and extension (data not shown) and is likely the result of a
minor rATP contamination in the rCTP preparation because the product
distribution is unchanged. The maximum length of primers that can be
synthesized with rCTP and rATP is five nucleotides and corresponds to
primers having the sequence 5
-AACCC. This is significant because
previous studies have demonstrated that elongation of RNA primers by
DNA pol
/primase requires a minimal length of seven to ten
nucleotides (19). Our results suggest that in fact DNA pol
can
recognize and extend RNA primers as short as five nucleotides.
Interestingly, the data presented in Figs. 3 and 4 reveal a distinct
product migrating at greater than 60 bases in all lanes. This product
is observed independent of rNTPs employed in the reaction and has also
been observed in some reactions without the addition of rNTPs. However,
this product was also observed in reactions employing a
[
-32P]rCTP label, suggesting that it is not a result
of DNA extension and labeling of the TS10 substrate via a stable
secondary structure (data not shown).
The results from this study identified a protein capable of
synthesizing the mammalian telomeric lagging strand in
vitro. The protein was identified as DNA pol /primase by
antibody inhibition experiments. Characterization of telomeric lagging
strand synthesis demonstrated complete dependence on rNTPs, suggesting
an RNA priming mechanism. Direct evidence of RNA priming of the TS10
substrate was obtained using a [
-32P]rCTP label (data
not shown). A unique primer initiation site was established, and
five-base-long ribonucleotide primers are capable of being extended
with DNA by pol
. Degradation of the RNA primers was accomplished by
RNase HI and FEN-1 consistent with their role in Okazaki fragment
processing.
The results presented suggest that DNA pol /primase has a role in
telomere maintenance. In a genetic screen to identify proteins that
interact with the yeast PRI1 gene, encoding the DNA primase subunit of pol
/primase, the MEC3 was isolated (22).
MEC3 has been found to participate in the G2
cell cycle checkpoint and, in conjunction with RAD24 and
RAD17, degrade the C-rich strand of telomeric and
subtelomeric DNA in response to DNA damage (23). The demonstration that
mec3,pri1 double mutants are synthetically lethal (22)
supports our hypothesis that primase is involved in mammalian telomere
maintenance. Interestingly, a recent study has identified the
Saccharomyces cerevisiae cdc13 gene product as a specific
telomere DNA binding protein (24). The finding that arrest of
cdc13 mutants requires RAD24 (25) and therefore MEC3 provides further evidence that primase is involved in
mammalian telomere metabolism.