* Department of Nephrology, Hypertension, and Genetics, Franz Volhard Clinic, Max Delbrück Center for Molecular Medicine,
Humboldt University, 13125 Berlin, Germany; and Department of Pediatrics and Department of Cell Biology, Harvard Medical
School, Boston, Massachusetts 02115
The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed "functional organization" of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1-28 and 111-179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins.
We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.
THE complexity of the mammalian genome and the
multitude of different biochemical processes occurring in the nucleus call for an efficient mechanism of
coordination and organization. At least in part this is
achieved by subdivision into functional domains (for review see 23, 54). DNA replication occurs at discrete nuclear foci (14, 18, 36, 37, 40, 58) where replication proteins
(proliferating cell nuclear antigen [PCNA],1 5; the 70-kD
subunit of replication protein A [RPA 70], 7; DNA polymerase One good candidate for localization at replication foci is
DNA ligase I (34). DNA ligase activity is required for
DNA replication, repair, and recombination. Yeast DNA
ligase I mutants are lethal and show defects in DNA replication and repair (19, 38). The human cDNA of DNA ligase I was cloned by functional complementation of yeast
conditional mutants and shows an open reading frame of 919 amino acids (2) and an active site lysine residue at position 568 (20). Mammalian DNA ligase I is essential for in
vitro replication of simian virus 40 DNA and cannot be
substituted by other DNA ligases (59). Inherited genetic
defects of the human DNA ligase I gene demonstrate its
requirement for Okazaki fragment ligation during lagging
strand DNA synthesis and for repair of DNA damage (3,
44, 53, 56), which is a prerequisite for genome integrity. Recent studies with homozygous null mice showed that in
less demanding selection conditions, DNA ligase I function can be substituted by another yet unknown ligase activity (4). The enzyme has a hydrophilic protease-sensitive
NH2-terminal domain of 249 amino acids (57), which has a
negative regulatory function that is relieved upon phosphorylation by casein kinase II (43). This NH2-terminal
domain is dispensable for enzyme activity in vitro, as well
as for complementation of yeast (2) and bacterial ligase mutants (20), though it is required in vivo in mammalian
cells (42). Moreover, this domain has no counterpart in the
recently identified human DNA ligases III and IV (8, 60),
despite extensive homology throughout their catalytic domains.
In this study we show that DNA ligase I is localized at
nuclear replication foci during S phase. We identified and
characterized a bipartite protein sequence that is necessary and sufficient for this cell cycle-dependent redistribution. Moreover, this sequence works position independently, can target heterologous proteins to subnuclear
sites of DNA replication and, therefore, meets the criteria
for a targeting sequence. We propose that this targeting sequence contributes to the high efficiency of Okazaki fragment ligation during lagging strand DNA synthesis and
plays an important role in the coordinate regulation of the
different steps of DNA replication. We furthermore used
fusions of DNA ligase I with GFP to directly visualize replication foci in vivo. The characteristic redistribution of
these fusion proteins makes them unique S phase markers
to monitor cell cycle progression in living cells.
Antibodies
Polyclonal antibodies against DNA ligase I were raised in rabbits using a
peptide antigen spanning amino acids 1-23 from the mouse enzyme with
the addition of N-chloracetyl-glycine at the NH2 terminus for coupling to
the carrier protein KLH (30). The anti-peptide antibody was purified
from serum by affinity chromatography as described by Sawin et al. (47)
using the peptide antigen coupled to Affi-Gel 10 (Bio Rad, Hercules, CA).
In addition, the following primary antibodies were used: mouse monoclonal anti-PCNA antibody (clone PC 10; Dako, Carpinteria, CA; Zymed,
San Francisco, CA), 12CA5 hybridoma supernatant recognizing the Flutag epitope, DNA Ligase I Fusion Constructs
Expression plasmids were derived from pJ3 Cell Culture and Transfection
Human HeLa cells, monkey Cos 7 cells, and mouse MEL and C3H10T1/2
cells were grown in a humidified incubator at 37°C and 5% CO2 in DME
supplemented with 10% fetal calf serum. Mouse (C2C12) and rat (L6E9)
myoblast cells were grown as above except that the media was supplemented with 20% fetal calf serum.
Cos 7 cells were transfected by the DEAE-dextran pretreatment
method as described by Leonhardt et al. (24). 2 d later, cells were scraped,
extracted, and analyzed as described below. Exponentially growing mouse
fibroblasts were transfected by the calcium phosphate-DNA coprecipitation method (15) with glycerol shock treatment (41) ~8-12 h later. 36-48 h
after DNA addition, cells were fixed and stained.
For visualization of GFP-ligase in living cells, transfected fibroblasts
plated onto glass-bottom petri dishes were changed to Hepes-buffered
medium (10 mM Hepes, pH 7.0, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Glucose) and screened with a fluorescence microscope
using an FITC filter.
Whole Cell Extracts and Western Blotting
Transfected Cos cells were scraped and extracted as described by Leonhardt et al. (24) in 0.2 M NaCl, 0.32 M sucrose, 0.3% Triton X-100, 20 mM
Tris-HCl (pH 7.4), 3 mM MgCl2, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonylfluoride. Cell extracts used to characterize the anti-DNA
ligase I antibody by immunoblots were made by extraction of cells for 30 min on ice with RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1%
NP-40, 0.5% deoxycholate, 0.1% SDS) containing the following protease
inhibitors: 1 mM EDTA, 1 mM Pefabloc, 10 µM leupeptin, 10 µM pepstatin,
and 10 µM aprotinin. After 5 min centrifugation in the cold, the soluble
fraction was analyzed by immunoblot. The protein extracts were dissolved
in Laemmli sample buffer, separated by SDS-PAGE under reducing conditions, and transferred to PVDF membranes. After incubation with epitope tag-specific antibodies or anti-DNA ligase I antibodies, followed by
HRP-conjugated secondary antibodies, the blots were developed using
the ECL detection procedure (Amersham, Buckinghamshire, UK).
Immunofluorescence
Transfected cells were washed in PBS and fixed for 10 min in 3.7% formaldehyde in PBS or for 5 min in cold methanol. All following incubations
were performed at room temperature. Formaldehyde-fixed cells were permeabilized for 10 min in 0.25% Triton X-100. Fixed cells were blocked in
5% goat serum or 0.2% gelatin and incubated for 60 min with the respective primary antibodies as described before (7, 24). After extensive washes
in 0.1% NP-40, cells were incubated for another 60 min in FITC-conjugated goat anti-mouse or anti-rabbit IgG and biotinylated goat anti-rabbit
or anti-mouse IgG antibodies, followed by washing and 30 min incubation
in streptavidin-Texas red, as described before (7). DNA was counterstained with Hoechst 33258 and cells were mounted in mowiol with 2.5%
DABCO (6). Pulse labeling with BrdU and detection of incorporated
BrdU were done as described before (7, 24).
Microscopy
Stained specimens or live cultures were examined and photographed in
microscopes (Axiophot and Axiovert; Zeiss, Inc., Thornwood, NY)
equipped with phase-contrast and epifluorescence optics, using 40, 63, and
100x oil immersion Plan-Neofluor and Planapochromat objectives. Single
Hoechst, FITC, and Texas red filters were used as well as FITC-Texas red
dual filters. Pictures were taken with Kodak Ektar and Royal Gold films.
Micrographs were scanned, assembled, and annotated with Adobe Photoshop and Canvas software in a Power MacIntosh computer and printed
with a Phaser 440 dye sublimation printer (Tektronix). Overlays were
mostly generated by photographic double exposure with the two respective filters or directly with a dual filter. In two cases (Fig. 2 C, the upper
left early S phase nucleus, and F), overlays were generated digitally using Adobe Photoshop layers.
Localization of DNA Ligase I throughout the Cell Cycle
To identify additional components of nuclear replication
foci and to probe the dynamics of nuclear architecture, we
analyzed the subcellular localization of DNA ligase I, which
is involved in joining Okazaki fragments during lagging-strand DNA synthesis (for review see 29).
Polyclonal antibodies were raised against mammalian
DNA ligase I. We used a peptide spanning the first 23 amino
acid residues of mouse DNA ligase I, which differs from
the human protein in two positions. The antisera were
then affinity purified and tested for specificity and species
reactivity by immunoblot. As shown in Fig. 1 A, both elution methods (0.2 M glycine-HCl, pH 2.0, and 6 M guanidine hydrochloride) yielded antibody fractions that specifically reacted with mouse DNA ligase I. The predicted size of mammalian DNA ligase I is 101-102 kD, but endogenous as well as recombinant proteins migrate anomalously
slowly in SDS-PAGE, which may be due to its high proline
content, to the presence of phosphoserine residues, and to
the high hydrophilicity of the NH2-terminal domain (43, 57).
The result obtained with our antibodies is in agreement
with the previously observed migration of mammalian DNA
ligase I. This signal is competed away by pre-incubating the antibodies with the peptide used as antigen but not with
a nonspecific peptide, as shown in Fig. 1 B, further confirming its specificity. Finally, we tested for cross-reactivity
with homologues from other mammalian species. Fig. 1 C
shows that the affinity-purified antibodies specifically recognize proteins of the expected size in extracts from mouse,
rat, monkey, and human cells. This result was expected because of the relatively high degree of conservation between human and mouse DNA ligase I cDNAs. Under the
conditions used (50 µg of extract) some additional signals can be seen in the monkey cell extracts, even though at
much lower intensity.
We then used the affinity-purified antibodies to analyze
the subcellular localization of DNA ligase I throughout
the cell cycle. Asynchronously growing mouse fibroblasts
and myoblasts were labeled with the thymidine analogue
BrdU for 5 to 10 min and immediately fixed, to detect subnuclear sites of DNA replication. BrdU incorporation was
visualized by staining with BrdU-specific mouse monoclonal antibodies, and samples were also stained for DNA
ligase I with the affinity-purified rabbit antibodies. The cells that did not incorporate BrdU, and are therefore in
G1 or G2 phase, exhibit a dispersed nucleoplasmic distribution of DNA ligase I with exclusion from nucleoli (data
not shown). As depicted in Fig. 2, A-C, the nuclei with a
punctate subnuclear BrdU pattern show colocalizing DNA
ligase-labeled foci, as can be better seen in the overlay of
the two images in C. This figure is a composite of nuclei, illustrating different patterns of subnuclear foci observed in
both mouse fibroblasts and myoblasts. We further investigated the distribution of DNA ligase in interphase cells by
costaining with anti-PCNA antibodies. One subnuclear
pattern is presented in Fig. 2, D-F, and clearly shows the
colocalization of both PCNA and DNA ligase I at sites of
DNA replication. Nuclei with disperse PCNA nucleoplasmic
signal, showed the same homogenous distribution of DNA
ligase I (data not shown). The DNA ligase I distribution was identical using formaldehyde and methanol fixation.
In mitotic cells, at the metaphase stage shown by the
alignment of the chromosomes in the metaphase plate in
Fig. 2 J, DNA ligase I is excluded from the condensed
chromosomes and distributes in the cytoplasm (Fig. 2 G).
Later, during chromatid separation and movement to the
spindle poles at anaphase in Fig. 2 K, DNA ligase I continues to be excluded from the condensed chromosomes (Fig.
2 H). At the end of telophase and cytokinesis, when the nuclear envelope reforms around the decondensing chromosomes (Fig. 2 L), DNA ligase I is immediately imported into the nucleus, as shown in Fig. 2 I.
In summary, DNA ligase I is excluded from condensed
chromosomes during mitosis and immediately reenters the
nucleus upon formation of the nuclear envelope. In interphase cells, it presents a homogenous nucleoplasmic pattern except during S phase, when it redistributes to subnuclear sites of DNA replication.
Mapping the DNA Ligase I Sequence Responsible for
Targeting to Replication Foci
Since there are no dividing membranes in the nucleus,
which could explain the concentration of proteins at these
foci, we decided to further investigate the basis for this cell
cycle-dependent redistribution. In the case of DNA MTase, a
distinct targeting sequence had been identified that is necessary and sufficient for association with replication foci
(24). The cDNA of human DNA ligase I has an open reading frame of 919 amino acids (2), and the protein resembles DNA MTase in that it also has a protease-sensitive NH2-terminal domain, in this case of 249 amino acids.
To test whether we could reproduce the endogenous
DNA ligase patterns with recombinant proteins, the enzyme was tagged by adding an epitope from the hemagglutinin of influenza virus (Flutag; Fig. 3 A) and visualized in
transfected cells by immunofluorescence microscopy. Double staining with antibodies against DNA MTase, which labels replication foci (24), showed co-localization of DNA ligase I at these sites (Fig. 3 B, a-c). Tagged DNA ligase I,
like the endogenous protein, redistributed during the cell cycle and showed the same patterns as DNA MTase and
PCNA (Fig. 3 B, and data not shown).
To investigate whether or not DNA ligase I uses a
mechanism similar to DNA MTase for association with
replication foci, a series of deletion mutants was generated
in search for a potential targeting sequence (Fig. 3 A). All
fusion proteins were tested for stability in vivo as described before (24). In brief, Cos cells were transfected
with the respective expression constructs; protein extracts
were made and tested by Western blot analysis (data not
shown). All constructs gave stable fusion proteins, which
argues against possible artefacts due to instability problems. The apparent molecular weight of the fusion proteins differed in most cases from values deduced from the
amino acid sequence. This discrepancy is consistent with the
observation that DNA ligase I itself migrates anomalously
slow (see Fig. 1). Two different epitope tags, Flutag and a
To fine map the region required for targeting, we constructed a series of COOH-terminal, NH2-terminal, and
internal deletions scanning the entire NH2-terminal regulatory domain. The structure of these fusion proteins and
their respective phenotype is shown in Fig. 3 A. Fig. 3 B,
d-f, illustrates one example of a targeting proficient deletion mutant. On the other hand, several deletions within
the regulatory domain abolished targeting, meaning the respective fusion proteins did not redistribute to replication foci in S phase nuclei and showed, instead, a dispersed
nucleoplasmic distribution (Fig. 3 B, g-h). The fine mapping identified a bipartite-targeting sequence, which encompasses amino acids 1-28 and 111-179. This bipartite
targeting sequence is necessary and sufficient to target heterologous proteins (like The regulatory domain also contains the NLS. We found
that the first 206 amino acids of DNA ligase I are sufficient
for nuclear localization, while fusion proteins containing
only the first 112 amino acids were clearly cytoplasmic
(data not shown). This finding indicates that nuclear localization of DNA ligase I requires sequences spanning amino
acids 112-206, which contains stretches of basic amino acids (e.g., amino acids 120-131 and/or 149-152) that fit the
description of a potential NLS (12, 50). A recent report
described the mapping of the DNA ligase I NLS to amino acids 119-131 (34), however, deletion of this region (removing amino acids 112-178 in the last ligase- Our results show that the classical NLSs (amino acids
120-131 and/or 149-152) are not absolutely required and
also not sufficient for nuclear localization and that other regions (amino acids 1-28 and 178-206) contribute to the
nuclear localization of DNA ligase I, which may be a combination of nuclear uptake and retention, as described for
other proteins (48).
The identification of the targeting sequence in the human DNA ligase I raised the question whether or not this
sequence was conserved in other proteins present at replication foci and in DNA ligases from other species. The
comparison with the DNA MTase targeting sequence (24)
shows no detectable similarity. On the contrary, both sequences are rather different; the DNA ligase targeting sequence is extremely hydrophilic, while the DNA MTase is
rather hydrophobic (Fig. 4 A). This difference makes sense in view of their different biological function and may simply reflect their targeting to different parts of the replication factories. That is, these various targeting sequences
most likely constitute protein-protein interaction surfaces
in vivo, which are as diverse as their binding partners.
A comparison with other ATP-dependent DNA ligases
shows that the catalytic domain is highly conserved throughout evolution; however, no sequences with homology to
the human targeting sequence were detected in lower eukaryotic or prokaryotic homologues (e.g., the comparison
with DNA ligase I from Schizosaccharomyces pombe, Fig.
4 B). On the other hand, the targeting sequence is conserved between human and mouse (72% identity; 46), and
the human targeting sequence also functions in mouse
cells (Figs. 3 B and 5 A). There is also considerable conservation (data not shown) with the Xenopus laevis DNA ligase I (25). The fact that the targeting sequence is missing
or is very different in lower eukaryotic and prokaryotic DNA ligases and is also not required for complementation
may indicate that it only recently developed in evolution.
Visualization of DNA Ligase I Subnuclear
Redistribution in Living Cells
So far, the existence of subnuclear replication foci has only
been shown by immunofluorescence. We therefore decided to directly probe these structures in living cells and
fused the full length as well as the NH2 terminus alone
containing the targeting sequence to the COOH-terminal
end of the GFP, as schematically drawn in Fig. 5. The chimeric proteins were expressed in mouse cells and observed live under the microscope using an FITC filter. In
addition to a nuclear dispersed distribution (first nucleus
on the left hand side of Fig. 5, A and B), several discrete
patterns of dots of different sizes and shapes were observed with the chimeric GFP-ligase proteins outlined in
Fig. 5. Representative examples of the latter patterns are
shown in the four nuclei at the right hand side of each
panel. These patterns in live cells match the early, mid,
and late S phase patterns reported in fixed and stained cells (14, 37, 40, 58). Based on that classification, the second nucleus from the left exhibits a fine punctate pattern of foci throughout the nucleoplasm characteristic of early
S phase. The nucleus in the middle shows a concentration
of foci at the nucleolar periphery corresponding to a later
S phase stage. The two nuclei at the right hand side have
larger foci with often irregular shape or loop-like structures, which are typical of late S phase. All these patterns
were also observed by staining fixed cells with anti-DNA
ligase I antibodies (Fig. 2) as well as with the other tagged
constructs (Fig. 3, and data not shown). These same GFP-ligase-expressing cells were fixed and stained with anti-PCNA and anti-MTase antibody (Cardoso, M.C., R. Reusch, and H. Leonhardt, unpublished results), confirming the
colocalization results presented in Figs. 2 and 3. The observation that the NH2-terminal domain of DNA ligase I
(which contains the targeting sequence mapped in Fig. 3)
fused at the COOH terminus of GFP (schematically shown
in Fig. 5 B) shows the same patterns as the full length DNA ligase I chimera with GFP indicate that the targeting
sequence works as a `module' and can function in the context of unrelated proteins and irrespective of its location in
the fusion construct. Furthermore, they allow direct visualization of subnuclear replication foci in living mammalian cells. The very characteristic redistribution of the GFP-ligase fusion proteins during the cell cycle makes these
constructs a unique S phase marker for studies in living cells.
In this study, we describe the subcellular localization of
DNA ligase I and analyze the basis for its cell cycle-dependent redistribution. We show that DNA ligase I is excluded from condensed chromosomes in mitotic cells and
is rapidly imported into the nucleus upon formation of the
nuclear envelope (Fig. 2). In interphase cells, DNA ligase I
is homogenously distributed throughout the nucleoplasm
except in S phase cells, when it is localized at subnuclear replication foci (Fig. 2). We mapped a bipartite targeting
sequence, which is necessary and sufficient for this S
phase-dependent redistribution (Fig. 3 A).
It should be mentioned that these results clearly differ
from a previous report (34). That report claimed the identification of a targeting sequence solely based on a single
deletion mutant not detected at replication foci, which, as
a negative result, is by definition, inconclusive. Deletions
often alter the overall folding and stability of proteins and
can unspecifically affect functions that are far apart in the
primary protein structure. For these reasons, a subcellular
targeting sequence is usually defined as a protein sequence
that (a) is necessary and (b) sufficient for subcellular localization; (c) works position independently; (d) is separated
from the catalytic domain, and (e) can target heterologous
proteins to the respective subcellular domain. In this study
we mapped the targeting sequence of DNA ligase I and demonstrated that this sequence by itself is sufficient to recruit different heterologous proteins to replication foci in
S phase cells. The targeting sequence functions as a module independently of its location in the fusion protein (see
Figs. 3 and 5) and deletion of this sequence in the context
of the entire DNA ligase I protein leaves the catalytic properties untouched (20) but abrogates its S phase-dependent
association with replication foci. We show that the DNA
ligase I targeting sequence is conserved between the human
and mouse enzymes, and the human sequence also targets
fusion proteins to replication foci in mouse cells (Fig. 3 B).
In lower eukaryotic homologues (e.g., yeast or Drosophila DNA ligases I) the NH2-terminal domain is also dispensable for enzyme activity in vitro (1, 45, 55). However,
these domains are clearly shorter and exhibit no similarity
among each other or to the higher eukaryotic counterparts
(Fig. 4 B, and data not shown). Furthermore, a truncated
human DNA ligase I protein, with the entire NH2-terminal
domain deleted, is able to rescue yeast DNA ligase I mutant strains (2) but cannot rescue homozygous null DNA
ligase I mutant mouse cells (42). The latter indicates that
the NH2-terminal domain of human DNA ligase I has essential functions in mammalian cells that are not required
in lower eukaryotes. The identification and mapping of the
targeting sequence (Fig. 3) now assigns a function to this
domain in vivo and provides a possible explanation for
those presumably conflicting in vitro and in vivo results.
Interestingly, the NH2-terminal regulatory domain is
also not conserved in the recently cloned mammalian
DNA ligases III and IV, despite a high degree of homology throughout the catalytic domains (8, 60). These two
enzymes are able to substitute for DNA ligase I in in vitro
DNA repair assays (22, 49) but not in replication assays
(31, 59). Altogether, these results suggest a function of the
NH2-terminal domain of DNA ligase I in DNA replication
rather than repair, which fits well with our mapping of the
targeting to replication foci in this domain. A comparison
with the previously identified targeting sequence of the
DNA MTase showed no discernible similarities (Fig. 4 A),
which suggests that these sequences interface with different components of the replication factory as illustrated in
Fig. 6. In this context it is interesting that biochemical fractionation experiments have shown that DNA ligase I is
present in megadalton, multiprotein complexes with multiple catalytic activities associated with DNA replication
(27, 28, 32, 39, 62). The identification of the targeting sequence now renders possible a directed search for interacting factors and the elucidation of the molecular architecture of these replication factories.
Similar targeting principles seem to apply also to other
functional domains of the mammalian nucleus (for review
see 23). Thus, targeting sequences were identified in the
Drosophila splicing regulators su(wa) and tra, which are
concentrated at distinct nuclear foci called "speckled compartment" (26). Further, the nucleolar localization of the
retroviral proteins HTLV-1 Rex (51), HIV-1 Tat (11, 13),
and HIV-1 Rev (9, 21) was shown to be mediated by nucleolar targeting sequences. Moreover, nucleolar targeting
of HIV-1 Rev was shown to be required for in vivo function (9, 21). Finally, the NH2-terminal 102 amino acids of
p80-coilin, a protein that localizes to nuclear coiled bodies,
are necessary and sufficient for targeting to this organelle
(63). All of these different sequences have one thing in
common: they are necessary and sufficient for targeting to
subnuclear domains; however, they do not share similar
protein motifs. On the contrary, they are as diverse as
their function and the domain to which they target.
The vast majority of studies on nuclear organization
have been done on fixed and stained cells, with the caveat
of possible artefacts (for review see 10). Several efforts
have been undertaken to use physiological buffers and unfixed cells to study subnuclear replication sites. Nevertheless, these studies were performed using permeabilized
cells that underwent still extensive manipulation before
these subnuclear compartments were visualized (16). Until now, localization of proteins at replication sites had not
yet been demonstrated in live cells. The localization of
DNA ligase I at replication foci (Fig. 2), the identification
of a bipartite targeting sequence in its NH2-terminal regulatory domain, which mediates cell cycle-specific association (Fig. 3), and the availability of the GFP allowed us to
address this issue directly. Chimeric proteins comprising
GFP and DNA ligase I (full length or NH2-terminal domain) allow the visualization of these subnuclear structures in living cells and show patterns indistinguishable
from fixed and stained cells (Fig. 5). These findings enable
direct visualization of S phase in living cells, which should
be very useful for cell cycle studies.
In summary, we describe the subcellular distribution of
DNA ligase I during the cell cycle and map the sequence
responsible for its association with replication foci in S
phase cells. We showed that the targeting sequence works
as an independent module and can direct unrelated proteins (like Altogether, these results suggest that targeting might be
a general principle of nuclear organization and might be a
means to cope with the growing complexity in higher eukaryotes as evolution progressed. The targeting sequence,
by bringing the catalytic domain to the right place at the
right time, allows catalysis to occur at higher order kinetics,
which meets the needs of a competitive and demanding
environment such as the mammalian nucleus. We propose
that the efficient coordination of complex processes such
as DNA replication, from controlled initiation to ligation of
Okazaki fragments and DNA methylation, might be accomplished in the mammalian nucleus by integration into assembly line-like protein factories (Fig. 6), which increase the
effective enzyme concentration, specificity, and processivity.
This dynamic nuclear architecture thus constitutes a higher
order mechanism of regulating enzyme activity in vivo.
, 17) are localized. In addition to these replication proteins, DNA methyltransferase (DNA MTase; 24) and
cell cycle proteins (cyclin A and cdk2; 7, 52) were also
found to redistribute to these foci during S phase. In view
of the complexity of mammalian DNA replication, it is
likely that many more proteins are localized at these nuclear foci.
Materials and Methods
-galactosidase (
-gal)-specific mouse monoclonal antibody
(Promega, Madison, WI), rabbit polyclonal anti-MTase antiserum (24),
and mouse monoclonal anti-5-bromo-2
-deoxyuridine (BrdU) antibody
FITC conjugated (Boehringer Mannheim, Mannheim, Germany).
(35) or pEVRF0 (33). The
latter was used to add a heterologous NLS (see below). Oligonucleotides
encoding a nine-amino acid epitope (YPYDVPDYA; 61) from the hemagglutinin of influenza virus (Flutag) were inserted at the Bsp EI restriction site (codon 307), at the Eag I restriction site (codon 773) or between
both sites in the cDNA of human DNA ligase I (ATCC 65856; 2). The
-gal
epitope was derived from the
-gal gene of Escherichia coli (amino acids
361-1,069) and was added at the COOH-terminus of all but two deletion
constructs. A short nuclear localization signal (NLS) derived from SV40
large T antigen (PKKKRKV) was added at the NH2 terminus of some deletion constructs to compensate for the loss of their own NLS using a translational fusion vector, as previously described (24). The green fluorescent protein (GFP) fusions were derived from the pRSGFP-C1 and the
pEGFPC1 vectors (Clontech, Palo Alto, CA), by inserting a fragment containing the full length or the first 250 amino acids of the human DNA ligase cDNA at the COOH terminus of the open reading frame of both
GFP mutants. It is noteworthy that the GFP expressed in these constructs,
with an additional 26 amino acids derived from the multiple cloning site
added to its COOH terminus, is by itself nuclear and cytoplasmic. Plasmid DNA was purified using columns (Qiagen, Hilden, Germany) according to the instructions of the manufacturer.
Fig. 2.
Localization of DNA ligase I throughout the cell cycle.
Asynchronously growing mouse fibroblasts (C3H10T1/2 cells;
A-C and G-L) and myoblasts (C2C12 cells; A-F) were pulse
labeled with BrdU for 5 to 10 min (A-C) and formaldehyde (A-C,
G, and J) or methanol fixed (D-F, H, I, K, and L). Cells were
stained for DNA ligase I with the affinity-purified anti-DNA ligase I rabbit antibodies (see Fig. 1 red; B, E, and G-I), for sites of
BrdU incorporation with anti-BrdU mouse monoclonal antibody
(green; A), and for PCNA with anti-PCNA-specific mouse monoclonal antibody (green; D), and DNA was visualized by counterstaining with Hoechst 33258 dye (J-L). A-F show the distribution
of DNA ligase I (B and E) in interphase cells relative to sites of
ongoing DNA replication labeled with BrdU (A) and to PCNA
(D), which has previously been shown to redistribute in S phase
nuclei to replication centers (5, 7). A-C is a composite of cell nuclei at different stages of S phase. As can be better visualized in
the overlay of the green and red images in C and F, DNA ligase I
takes on a pattern of subnuclear foci that colocalize with sites of
BrdU incorporation (C) and with PCNA (F). G-L show the distribution of DNA ligase I in mitotic cells. A cell in metaphase as
evidenced by the absence of nuclear membrane and the alignment of the chromosomes in the metaphase plate in J, shows that
DNA ligase I is excluded from the condensed chromosomes and
upon nuclear envelope breakdown distributes in the cytoplasm
(G). During chromatid separation and movement to the spindle
poles at anaphase in K, DNA ligase I is still dispersed in the cytoplasm and excluded from the condensed chromosomes (H). At
the end of telophase and cytokinesis, when the nuclear envelope
reforms around the decondensing chromosomes (L), there is an
immediate import of DNA ligase I into the nucleus as seen in I. Bars, 10 µm.
[View Larger Version of this Image (43K GIF file)]
Fig. 1.
Purification and characterization of antibodies against
DNA ligase I. (a) Immunoblot analysis of MEL whole cell extract
(20 µg/lane) with anti-DNA ligase I antibodies. Lane 1, Preimmune serum; lane 2, affinity-purified antibodies eluted with 0.2 M glycine-HCl, pH 2.0; lane 3, affinity-purified antibodies eluted with 6 M guanidine hydrochloride. A band with an apparent molecular weight of 120 to 130 kD reacts specifically with the affinity-purified antibodies. (b) Specifity of the antibody (lane 1) was
further tested by pre-incubation with specific (S, lane 2) and unspecific (U, lane 3) peptides at a 100-fold molar excess. The DNA
ligase I signal is competed out only with the NH2-terminal DNA
ligase I peptide and not with the same amount of an unrelated
peptide, confirming the specificity of the antibody. (c) Species reactivity of anti-DNA ligase I antibodies: lane 1, C2C12 (mouse)
cell extract; lane 2, L6E9 (rat) cell extract; lane 3, Cos 7 (monkey)
cell extract; lane 4, HeLa (human) cell extract. A total of 50 µg of
whole cell extracts was loaded in each lane. The anti-DNA ligase
I antibodies specifically detect a protein band of similar size in rat
and mouse cell extracts and slightly bigger in monkey and human
cell extracts, which correspond most likely to the DNA ligase I
protein from these species.
[View Larger Version of this Image (32K GIF file)]
Results
Fig. 3.
Mapping of the human DNA ligase I targeting sequence. (A) Sixteen different epitope-tagged deletion mutations
of DNA ligase I were constructed. Their structure is schematically outlined, and their respective capability to associate with
nuclear replication foci is indicated with + (targeting proficient)
and (targeting deficient). Numbers on the left refer to the
amino acids of human DNA ligase I remaining in the deletion
constructs. The structure of DNA ligase I is outlined on the top,
showing the location of the regulatory NH2-terminal domain,
which is dispensable for enzyme activity in vitro (43), and the position of the active site lysine residue 568 (20). Notice that the
lower part of the graph is an enlargement of the first 263 amino
acids to better display the results of the fine mapping. Shaded
boxes highlight the bipartite targeting sequence that is necessary
and sufficient for association with replication foci, as defined by
these deletion constructs. As indicated, the first four constructs
are full length or deletion mutants of DNA ligase I tagged with
the Flutag epitope inserted at codons 307, 773, or between both.
A representative example of one S phase pattern of these Flu-tagged proteins is shown in part (B, a-c). The following 12 DNA
ligase I deletion mutants are fused at their COOH terminus to
-gal
(amino acids 361-1,069) as described in 24. The
-gal part of the
fusion proteins is not depicted again in the lower half of this
graph. The black diamond represents a short NLS derived from
SV40 large T antigen and was added at the NH2 terminus of some
deletion constructs to compensate for the potential loss of their
own NLS using a translational fusion vector. The expression of all
listed fusion proteins was monitored by Western blot analysis
(data not shown). (B) The subnuclear patterns of the Flu epitope-tagged and
-gal fusion proteins with human DNA ligase I were
determined by transiently expressing the fusion construct into
mouse C3H10T1/2 cells and double staining the formaldehyde-fixed cells for: DNA ligase I (red; a, d, and g) using an epitope-specific monoclonal antibody; DNA MTase (which redistributes
to replication sites during S phase) using rabbit polyclonal antiserum (green; b, e, and h); and overlay of DNA ligase and DNA
MTase staining (c, f, and i). a-c depict a nucleus of a cell transfected with full length DNA ligase I tagged at codon 773 with the
Flu epitope (corresponding to the first construct in A) in late S
phase, and the double exposure in c shows the colocalization of
DNA ligase I and DNA MTase at nuclear replication foci. Both
proteins show similar redistribution within the nucleus during the
cell cycle, which also parallels the one of PCNA, as shown in Fig.
2. d-f show one example of a targeting-proficient fusion construct
with
-gal (d; containing amino acids 1-28 and 111-263 of DNA
ligase I) that colocalizes with DNA MTase (e) at replication foci
as can be seen in the double exposure (f). g-i show a targeting-
deficient fusion construct with
-gal (g; containing amino acids
62-212 of DNA ligase I) that takes on a dispersed distribution
and does not redistribute during S phase to replication foci as visualized with DNA MTase antibodies (h) and also in the double
exposure (i). Bars, 10 µm.
[View Larger Versions of these Images (27 + 35K GIF file)]
-gal-derived epitope, were inserted at different positions
in the DNA ligase I cDNA, to rule out potential tagging
artefacts. Two mutations deleting amino acids 308-772 and
264-919, immediately indicated that the entire catalytic
domain was dispensable for association with replication
foci. The latter were visualized as in Fig. 3 B, a-c, or by costaining with PCNA-specific antibodies (data not shown).
-gal from Escherichia coli) to
replication foci, since deletion of either part eliminates targeting (Fig. 3 A). Furthermore, this is the only targeting
sequence within DNA ligase I, since deletion of just the
first 28 amino acids in the context of the full length protein, abrogates targeting (Fig. 3 A). In other words, the
catalytic domain (amino acids 250-919) by itself does not
localize at replication foci, emphasizing the modular arrangement of targeting sequence and catalytic domain. While this work was in progress, a study geared towards
the mapping of the NLS of DNA ligase I was published
(34). In that report, one deletion construct lacking the first
115 amino acids was analyzed for subnuclear localization
and found to be absent from replication foci, which is consistent with our mapping results.
-gal fusion
depicted in Fig. 3 A) does reduce but not abolish nuclear
localization, i.e., the overproduced recombinant protein
can be found in the nucleus and cytoplasm (data not
shown). On the other hand, deletion of the first 28 amino
acids also affected nuclear localization. These discrepancies with the previous study (34) can in part be explained because their experiments were based on fusions to a reporter protein that by itself can enter the nucleus.
Fig. 4.
(A) Comparison between the targeting sequences of
DNA ligase I and of DNA MTase. A hydrophilicity plot was prepared for both enzymes, and the respective targeting sequences
were highlighted by shaded boxes. The overall structure of both
enzymes was outlined by delineating the respective regulatory
and catalytic domains and indicating the position of the active
site residues. The targeting sequences do not share any sequence
homology and are on the contrary very different; the bipartite
targeting sequence of the DNA ligase I is extremely hydrophilic,
while the DNA MTase sequence falls into a rather hydrophobic
domain. In both cases the targeting sequence is located in the
protease-sensitive, regulatory domain and is dispensable for enzyme activity in vitro. (B) Mammalian DNA ligase-targeting sequence is not conserved in lower eukaryotic homologues. The
amino acid sequence of the human DNA ligase I was compared
with the Schizosaccharomyces pombe homologue using the DNA
Strider program version 1.2 (C. Marck) and the results are displayed in a dot plot format. The yeast and human enzymes show
a high degree of homology throughout the catalytic domain, which
is outlined in the graph; however, no homologous sequences for
the NH2-terminal domain of the human enzyme (including the targeting sequence) could be detected in the fission yeast enzyme. Similar results are obtained comparing the human to the
budding yeast protein.
[View Larger Version of this Image (30K GIF file)]
Fig. 5.
Visualization of DNA ligase I subnuclear localization
in living cells. Asynchronous populations of mouse fibroblast and
myoblast cells were transfected with plasmid DNA containing the
full length (A) and the NH2-terminal 250 amino acids (B) of human DNA ligase I fused at the COOH terminus of the GFP. One
day after DNA addition, cells were split onto glass bottom petri
dishes, and the following days media was changed for a Hepes-buffered media. Cells expressing GFP-ligase fusion were screened
under the microscope using an FITC filter and photographed.
Below the micrographs are the respective schematic representations of the GFP fusion proteins with the full length human DNA
ligase I (A) and with the NH2-terminal 250 amino acids of human
DNA ligase I (B) containing the targeting sequence responsible
for association with replication foci during S phase. The regulatory and catalytic domains of DNA ligase I are depicted, and
MCS stands for multiple cloning site, which provides appropriate
restriction sites for translational fusions. Bars, 10 µm.
[View Larger Version of this Image (66K GIF file)]
Discussion
Fig. 6.
Model of a DNA replication and methylation factory.
The DNA double strand (thick lines) is spooled through a multiprotein complex, often referred to as "replication factory," which
is attached to the nuclear matrix (17). The newly synthesized
strands are represented by thin lines, and interruptions represent
Okazaki fragments of the lagging strand. The numerous participating enzymes in these factories (only two are depicted) are organized in an assembly line-like fashion, which ensures that, upon
passage through these factories, DNA is fully replicated, all Okazaki fragments are ligated, and all methyl groups (CH3) are
added to the new strand at hemimethylated sites. This organization is in part achieved by the tethering of DNA ligase I and DNA
MTase to the respective sites of these factories via the targeting
sequences mapped in these enzymes (see Fig. 3 and reference
24). Targeting sequences are depicted as separate domains since,
in both cases (DNA ligase I and DNA MTase), they are protease-sensitive domains, dispensable for enzyme activity in vitro and they
are necessary and sufficient for localization at replication foci.
[View Larger Version of this Image (84K GIF file)]
-gal from Escherichia coli and GFP from Aequorea victoria) to sites of DNA replication irrespective of
its position in the fusion protein. The targeting sequence
works across species (human and mouse) as well as in different cell types.
Received for publication 13 February 1997 and in revised form 15 August 1997.
Address all correspondence to Heinrich Leonhardt, Franz Volhard Clinic, Department of Nephrology, Hypertension, and Genetics, Wiltbergstrasse 50, 13125 Berlin, Germany. Tel.: (30) 9417-2341. Fax: (30) 9417-2336. E-mail: hleon{at}mdc-berlin.deWe are grateful to F.C. Luft and H. Haller for encouragement, support, and critical reading of the manuscript. We are indebted to M. Cochran, C. Clark, and L. Steinberg for their very special support of this work, and we would also like to acknowledge D. Hänlein for untiring help and advice with our computers. We thank G. Vargas for constructing a GFP-DNA ligase fusion.
This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Le 721/2-1) and from the Max Delbrück Center for Molecular Medicine.
-gal,
-galactosidase;
GFP, green
fluorescent protein;
MTase, methyltransferase;
NLS, nuclear localization
sequence;
PCNA, proliferating cell nuclear antigen.
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