(Received for publication, December 3, 1996)
From the Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom
The joining of Okazaki fragments during lagging strand DNA replication in mammalian cells is believed to be due to DNA ligase I. This enzyme is composed of a 78-kDa carboxyl-terminal catalytic domain and a 24-kDa amino-terminal region that is not required for ligation activity in vitro. Extracts of the human cell line 46BR.1G1, in which DNA ligase I is mutationally altered, supported aberrant in vitro SV40 DNA replication; the joining of Okazaki fragments was defective, and unligated intermediates were unstable. Human DNA ligase I, but not DNA ligase III or bacteriophage T4 DNA ligase, complemented both defects in 46BR.1G1 extracts. The catalytic domain of DNA ligase I was 10-fold less effective in complementation experiments than the full-length protein, indicating that the amino-terminal region of the enzyme is required for efficient lagging strand DNA replication. Moreover, in vitro SV40 DNA replication in normal human cell extracts was inhibited by an excess of either full-length DNA ligase I or the amino-terminal region of the protein, but not by the catalytic domain. This inhibition may be mediated by the interaction of the amino-terminal region of DNA ligase I with other replication proteins.
Several distinct DNA ligases have been identified in mammalian
cells, DNA ligase I being a major activity in proliferating cells (1,
2). Cytostaining experiments with antibodies against DNA ligase I
showed that the enzyme is specifically localized in the nucleus with
the same granular staining pattern as DNA polymerase , implicating
DNA ligase I in DNA replication (3). DNA ligase I (in conjunction with
DNA polymerase
,
, or
; RNase H1; and the 5
-nuclease DNase
IV/FEN-1) is able to complete lagging strand DNA replication in
vitro on a synthetic DNA substrate (4), and DNA ligase I activity
also functions to generate closed circular DNA during SV40 DNA
replication reconstituted with SV40 large T antigen and purified
mammalian proteins (5, 6).
The human DNA ligase I cDNA encodes a 102-kDa polypeptide (7). A 78-kDa carboxyl-terminal domain shows significant amino acid sequence homology to the CDC9 and cdc17+ gene products of Saccharomyces cerevisiae and Schizosaccharomyces pombe as well as to the human DNA ligase III and IV cDNAs. This domain is catalytically active in vitro in the absence of the amino-terminal region and is able to complement a conditional/lethal DNA ligase mutant of Escherichia coli (8). The 24-kDa amino-terminal portion of human DNA ligase I is a protease-sensitive hydrophilic region that has no counterpart in other mammalian DNA ligases or the yeast DNA ligases. Although this amino-terminal region is not required for activity of DNA ligase I in standard in vitro DNA joining assays, it is essential in vivo to counteract the lethal effect of knocking out DNA ligase I in mouse embryonic stem cells by the ectopic expression of DNA ligase I (9). A functional role for the amino-terminal portion of DNA ligase I during DNA replication has not been directly demonstrated, but this region may serve to localize the protein to sites of DNA replication by specific contacts with other replication factors (10).
The human cell line 46BR, derived from an individual exhibiting
retarded growth, severe immunodeficiency, and lymphoma, has been shown
to have an inactivating Glu-566 Lys mutation in one allele of the
DNA ligase I gene and an Arg-771
Trp mutation in the other (11).
Both of these amino acid changes are within the catalytic domain of the
protein. The SV40-transformed subline 46BR.1G1 is either homozygous or
hemizygous for the mutation at Arg-771 and shows the same physiological
defects as the primary cell line, including the accumulation of low
molecular size DNA species during DNA replication (12). 46BR.1G1 cells
contain normal levels of DNA ligase I protein, but the protein exhibits only ~5% of normal DNA joining activity, and the cells show delayed joining of Okazaki fragments during DNA replication in permeabilized, synchronized cells (13).
In this study, we have used the in vitro SV40 DNA replication system to monitor aberrant DNA replication in 46BR.1G1 cell-free extracts. This permeable system allowed us to specifically study the function of DNA ligase I and the amino-terminal region of the enzyme in DNA replication. We show that DNA ligase I, but not DNA ligase III or bacteriophage T4 DNA ligase, complements the replication defect in 46BR.1G1 cell-free extracts and that the amino terminus of DNA ligase I is required for efficient complementation. Furthermore, the addition of DNA ligase I to normal cell extracts, in >10-fold excess over the amount required for complementation of 46BR.1G1 extracts, inhibits their ability to perform in vitro SV40 DNA replication, and this inhibition appears to be mediated by the amino terminus of the protein.
Preparation of Cell-free Extracts
The human SV40-transformed fibroblast cell lines MRC5 V1 and 46BR.1G1 (13) were maintained in monolayer culture in E4 medium supplemented with 10% fetal bovine serum. Human HeLa S3 cells, adapted for growth in suspension (14), were grown in RPMI 1640 medium with 5% fetal bovine serum. Cells (1-5 × 109) were harvested in mid-log phase by centrifugation at 1000 × g for 5 min. Extracts were prepared from each cell line according to Cecotti et al. (14).
Reagent Enzymes
SV40 large T antigen (TAg)1 was obtained from Molecular Biology Resources, Inc. (Milwaukee, WI), and T4 DNA ligase was from New England Biolabs Inc. Recombinant human DNA ligase III and XRCC1 proteins were produced as described (15).
Subcloning, Expression, and Purification of Recombinant DNA Ligase I Proteins
Hlig I-(2-263)The DNA sequence encoding the
amino-terminal region of human DNA ligase I was amplified by polymerase
chain reaction using a 5-primer that included an NdeI
restriction site and a sequence encoding Met-Gly-His6
immediately upstream of the second residue of the amino-terminal
sequence of DNA ligase I. The polymerase chain reaction product was
cleaved with NdeI and at an internal BamHI
restriction site, cloned between the NdeI and
BamHI sites of pET11a, and verified by DNA sequencing. The
resultant construct encoded amino acids 2-263 of the full-length
protein.
Digestion of the DNA ligase I cDNA at
the internal BamHI site and at an AvrII site in
the 3-untranslated region generated a fragment that lacked the first
261 amino acids of DNA ligase I and that could be cloned into the
BamHI and CelII restriction sites of pET16b,
using an AvrII-CelII linker at the 3
-end, to generate an open reading frame encoding
Met-Gly-His10-Ser-Ser-Gly-His-Ile-Glu-Gly-Arg-His-Met-Leu-Glu from the vector polylinker, followed by the C-terminal domain of DNA
ligase I beginning at residue 262.
The AvrII-CelII linker used
above also contained a BamHI site, so the carboxyl terminus
of DNA ligase I could be isolated from Hlig I-(262-919) as a
BamHI fragment and joined in frame to the amino terminus in
pET11a by ligation to BamHI-linearized Hlig I-(2-263). This
generated a construct encoding histidine-tagged full-length DNA ligase
I. Protein expressed from this construct was catalytically active in
the absence of post-translational modification, in contrast to DNA
ligase I expressed previously as a -galactosidase fusion protein,
which required phosphorylation by casein kinase II for full catalytic
activity (16, 17).
All three constructs were transformed into E. coli strain
BL21. Individual colonies were grown to mid-log phase in L-broth supplemented with 50 µg/ml carbenicillin and then induced with 100 µM isopropyl--D-thiogalactopyranoside for
4 h at 20 °C. Cells were harvested by centrifugation at
1000 × g for 5 min and washed once in
phosphate-buffered saline, and the pellets were stored at
80 °C.
Pellets were rapidly thawed and resuspended in buffer A (50 mM Hepes-NaOH, pH 8.0, 500 mM NaCl, 0.1 mM EDTA, and 10% glycerol) supplemented with 1 mM DTT and protease inhibitors. Cells were lysed by
sonication, and debris was removed by centrifugation at 14,000 × g for 20 min. Sonic extracts were loaded onto a column of
Ni2+-nitrilotriacetic acid resin (QIAGEN Inc.) equilibrated
with buffer A. The column was washed with buffer A containing 1 mM imidazole and 1 mM DTT and then step-eluted
with increasing concentrations of imidazole in buffer B (50 mM Hepes-NaOH, pH 7.0, 100 mM NaCl, 0.1 mM EDTA, 10% glycerol, and 1 mM DTT). Hlig I
was eluted with 80 mM imidazole, Hlig I-(2-263) with 150 mM imidazole, and Hlig I-(262-919) with 250 mM
imidazole. Hlig I-(2-263) was further purified on an S-Sepharose
column equilibrated with buffer B containing 1 mM EDTA and
eluted in the same buffer containing 600 mM NaCl. All three
proteins were dialyzed against 20 mM Hepes-KOH, pH 7.5, 30 mM NaCl, 1 mM EDTA, 1 mM DTT, and
10% glycerol and stored at
80 °C.
DNA Ligase Assays
DNA ligation activity was measured by the conversion of
[5-32P]oligo(dT)16 annealed to poly(dA) to
multimers of [5
-32P]oligo(dT)16 at 37 °C
as described previously (13).
Pulse-Chase in Vitro SV40 DNA Replication Assay
Pulse-chase replication conditions were modified from the
procedure of Bullock et al. (18). Reaction mixtures (70-120
µl) contained 30 mM Hepes-KOH, pH 7.5, 7 mM
MgCl2, 0.5 mM DTT, 4 mM ATP, 40 mM creatine phosphate, 25 µg/ml creatine phosphokinase, 6 µg/ml superhelical replicative form I plasmid DNA containing an SV40
origin of replication, 4.8 mg/ml cell extract, and 40 µg/ml TAg.
Where reactions were supplemented with recombinant DNA ligase proteins,
the protein was preincubated with cell extract for 12 min at 37 °C.
Reaction mixtures were incubated for 20 min at 37 °C in the absence
of TAg to reduce TAg-independent background labeling of contaminating
replicative form II DNA and then incubated for 30 min at 37 °C in
the presence of TAg. Reactions were pulse-labeled for 20 s by the
addition of CTP, GTP, and UTP to 200 µM each; dCTP, dGTP,
and dTTP to 100 µM each; and 40 µCi/ml
[-32P]dATP (3000 Ci/mM; Amersham Corp.) to
give a final dATP concentration of 0.013 µM. Reactions
were chased by the addition of unlabeled dATP to 5 mM,
followed by incubation for various lengths of time at 37 °C. 25-µl
aliquots were stopped on ice with the addition of EDTA to 20 mM. 5 µl of each sample was removed for quantification of
32P radiolabel incorporated from
[
-32P]dATP into DNA (see below). The remaining 20 µl
was brought to 0.5% SDS and digested with 200 µg/ml proteinase K
(Sigma) at 37 °C for 1 h. After extraction once with
phenol/chloroform/isoamyl alcohol (24:24:1), the samples were
precipitated with 2 volumes of ethanol in the presence of 2.5 M ammonium acetate. After centrifugation, the pellets were
washed with 70% ethanol, dried, and resuspended in distilled water
supplemented with 20 µg/ml RNase A (Sigma) at 20 °C for 1 h
before the addition of 0.33 volume of alkaline gel loading buffer (200 mM NaOH, 4 mM EDTA, 10% Ficoll, and 0.1% bromcresol green). Samples were electrophoresed through a 1% alkaline agarose gel (19) at 2 V/cm for 16 h. Gels were fixed in 7% (w/v) trichloroacetic acid, dried, and autoradiographed.
In Vitro SV40 DNA Replication Assay
The assay conditions were slightly modified from the procedure
of Li and Kelly (20). Reaction mixtures (50 µl) contained 30 mM Hepes-KOH, pH 7.5, 7 mM MgCl2,
0.5 mM DTT, 4 mM ATP, 200 µM CTP,
µM GTP, µM UTP, 100 µM dCTP,
µM dGTP, µM dTTP, 30 µM dATP, 2 µCi of [-32P]dATP, 40 mM creatine
phosphate, 1.2 µg of creatine phosphokinase, 0.3 µg of superhelical
replicative form I plasmid DNA containing an SV40 origin of
replication, 240 µg of cell extract, and 2 µg of TAg. Reactions
were incubated at 37 °C for 2 h, and the extent of DNA
replication was monitored by quantifying the incorporation of
32P radiolabel into DNA (see below). Where assays were
supplemented with recombinant DNA ligase I proteins, the protein was
preincubated with cell extract for 12 min at 37 °C before the
extract was used in the assay.
Quantification of Radiolabel Incorporated from
[-32P]dATP into DNA
Aliquots of reaction mixtures were spotted onto DE81 filters
(Whatman), washed (3 × 15 min) in 200 ml of 0.5 M
Na2HPO4 to remove unincorporated
[-32P]dATP, rinsed in water and then in ethanol, and
air-dried, and incorporation of radioactive material into DNA was
determined by liquid scintillation counting.
The SV40-transformed human
fibroblast cell line 46BR.1G1 has ~5% of normal DNA ligase I
activity. Previous studies with permeabilized, synchronized cells
showed that ligation of Okazaki fragments is retarded in 46BR.1G1 cells
(13). Extracts from both 46BR.1G1 cells and a control SV40-transformed
fibroblast cell line, MRC5 V1, are able to support in vitro
SV40 DNA replication to similar extents, as estimated by incorporation
of 32P from [-32P]dATP into plasmid DNA
containing an SV40 origin of replication (data not shown). We have used
pulse-chase experiments to specifically monitor the maturation of
Okazaki fragments during in vitro SV40 DNA replication in
these extracts. Origin-containing plasmid DNA was preincubated in a
replication reaction mixture containing ATP but lacking other rNTPs and
dNTPs to allow origin complex formation, prior to pulse labeling with a
mixture of rNTPs, dNTPs, and [
-32P]dATP and then
chasing for various lengths of time with an excess of unlabeled
dATP.
Analysis of replication products on denaturing alkaline agarose gels
revealed that during the initial 20-s pulse, the majority of
32P incorporation from [-32P]dATP in both
MRC5 V1 and 46BR.1G1 extracts was into DNA fragments that migrated
between the 125- and 564-nucleotide markers, which corresponds to the
expected size of Okazaki fragments (Fig. 1A, lanes 5 and 9). However, in MRC5 V1 extracts,
larger DNA molecules were present even at this early time point, and
with increasing incubation times after the addition of unlabeled dATP,
pulse-labeled products were chased into higher molecular size forms
(Fig. 1A, lanes 6-8). After 15 min,
pulse-labeled products included molecules approaching 6.5 kilobase
pairs in length, the size of the substrate plasmid, and often molecules
migrating more slowly than the full-length plasmid, which may represent
fully replicated plasmids that were not yet decatenated. In contrast,
pulse-labeled products in 46BR.1G1 extracts were resolved into two
distinct species: small molecular size fragments, which appeared to be
unligated Okazaki fragments, and larger fragments, which probably
corresponded to the advancing leading strand (Fig. 1A,
lanes 10-12). Even after 15 min, the majority of
radiolabeled Okazaki fragments remained unligated in 46BR.1G1 extracts.
Furthermore, the larger pulse-labeled products were only approximately
half the length of those seen in MRC5 V1 extracts, and no products
larger than the full-length plasmid were observed. Both 46BR.1G1 and
MRC5 V1 extracts also showed a small amount of incorporation into forms
I and II of the plasmid, which could be seen in the absence of TAg
(Fig. 1A, lanes 1-4) and probably represents
background nick translation activity in a contaminating fraction of the
substrate plasmid that is initially present as form II. The background
incorporation was consistently 3-4-fold higher in 46BR.1G1 extracts,
and this most likely reflects more extensive nick translation in this
DNA ligase I-deficient cell line.
Unligated Pulse-labeled Replication Products Are Unstable in 46BR.1G1 Extracts
The amount of radioactivity incorporated into replication products in 46BR.1G1 extracts during the pulse labeling appeared to decrease throughout the subsequent chase period (Fig. 1A, lanes 9-12), indicating that such partial replication products are unstable in 46BR.1G1 extracts. Incorporation of radiolabeled material into DNA during pulse-chase assays was quantitated by adsorption of samples to DE81 filters, followed by scintillation counting. In MRC5 V1 extracts, as expected, there was no further detectable incorporation of radioactive material into DNA during the chase period (Fig. 1B). However, in 46BR.1G1 extracts, the amount of radioactive material already incorporated into DNA during the pulse actually decreased by >50% during the chase period. Pulse-labeled fragments of decreased size were not detectable during the chase period. In the absence of DNA ligation, nick translation may occur at the junction between adjacent pulse-labeled products and could account for the diminishing signal during the chase period.
Overexpression of Full-length DNA Ligase I and Fragments of the Recombinant ProteinHuman DNA ligase I has a 78-kDa
carboxyl-terminal catalytic domain and a 24-kDa amino-terminal region
that is not required for catalytic activity in vitro (8). To
assess the role of the amino-terminal region in DNA replication, we
made use of a BamHI restriction site in the cDNA of
human DNA ligase I that traverses codon 262 to subclone the individual
amino-terminal (Hlig I-(2-263)) and carboxyl-terminal (Hlig
I-(262-919)) domains (Fig. 2A). Proteins
were overproduced in E. coli with amino-terminal histidine
tags to allow rapid affinity purification on
Ni2+-nitrilotriacetic acid-agarose (Fig. 2B).
The ligation activity of full-length DNA ligase I and Hlig I-(262-919)
was measured against 1 unit of T4 DNA ligase on a double-stranded
substrate of [5-32P]oligo(dT)16 annealed to
poly(dA). Ligation activity generates [5
-32P]oligo(dT)16 multimers, which are
resolved on denaturing polyacrylamide gels (Fig. 2C).
Full-length DNA ligase I and Hlig I-(262-919) had comparable DNA
joining activity on this substrate (1.2 and 1.7 units/pmol,
respectively). Previously, up to 249 amino acids had been removed from
the amino terminus of DNA ligase I without loss of catalytic activity
(8), so Hlig I-(262-919) redefines the minimal catalytic domain of the
protein.
Complementation of the Replication Defect in 46BR.1G1 Extracts with Recombinant DNA Ligase I Is Promoted by the Amino-terminal Region of the Protein
Pulse-chase assays in 46BR.1G1 extracts were
supplemented with full-length recombinant DNA ligase I in an attempt to
complement the replication defect. In pulse-labeling experiments with
chase periods of 0 and 5 min, 0.02 units/µl full-length DNA ligase I was found to restore joining of Okazaki fragments in 46BR.1G1 extracts
to a level comparable to that seen in unsupplemented MRC5 V1 extracts
(Fig. 3A, lanes 5-8). The
addition of up to 0.2 units/µl enzyme did not further increase the
rate of ligation in 46BR.1G1 extracts (Fig. 3A, lanes
9 and 10) and had no effect on replication in MRC5 V1
extracts (lanes 1-4), suggesting that DNA ligase I is not
limiting in MRC5 V1 extracts. Supplementing 46BR.1G1 extracts with DNA
ligase I not only restored the chasing of Okazaki fragments into larger
DNA species, but also completely prevented instability of the
pulse-labeled DNA fragments during the chase period (data not shown).
Complementation also increased the level of incorporation into form I
DNA observed after the 5-min chase (Fig. 3A, lanes 8 and 10). This appears to correspond to the synthesis of
mature replication products, as background incorporation of radiolabel
into forms I and II of the plasmid by nick translation activity was
reduced in complemented 46BR.1G1 extracts (Fig. 3A,
lanes 7 and 9).
To establish whether the amino-terminal region of DNA ligase I is required for the replication function of the enzyme, analogous experiments were performed using Hlig I-(262-919), which lacks the amino-terminal part of the protein and encodes only the carboxyl-terminal catalytic domain. Supplementing assays with this protein at 0.02 units/µl had no effect on the joining of Okazaki fragments in 46BR.1G1 extracts, even though this amount of the full-length protein was able to fully complement defective joining (Fig. 3B, lanes 1-4). However, the addition of 0.2 units/µl Hlig I-(262-919) did complement the joining defect in 46BR.1G1 extracts, and again, stabilization of pulse-labeled products was simultaneously achieved (Fig. 3B, lanes 5 and 6). The percentage of labeled replication products smaller than the 564-nucleotide marker that remained after a 5-min chase was calculated from the data in Fig. 3 (A and B) and from further experiments in which full-length DNA ligase I or the catalytic domain was added in limiting amounts to ascertain the minimum necessary to achieve complementation (data not shown). The catalytic domain of DNA ligase I was only effective when added at 10-fold excess over the amount of full-length protein required for complementation (Fig. 3C), suggesting that although the amino-terminal region is not essential for the replication activity of DNA ligase I, it greatly improves the efficiency of the reaction.
Human DNA Ligase III and T4 DNA Ligase Cannot Substitute for DNA Ligase I during DNA Replication in 46BR.1G1 ExtractsBoth DNA
ligases I and III are major DNA joining activities in mammalian
fibroblasts (21); therefore, the ability of recombinant human DNA
ligase III to complement the replication defect in 46BR.1G1 extracts
was examined. The effectiveness of the reagent enzyme T4 DNA ligase in
this system was also analyzed. In experiments identical to those using
recombinant DNA ligase I, neither human DNA ligase III nor T4 DNA
ligase was able to significantly stimulate ligation of replication
products in 46BR.1G1 extracts after a chase period of 5 min (Fig.
4), even though DNA ligase III and T4 DNA ligase were in
3- and 10-fold excess, respectively, over the level (in units of DNA
joining activity) of DNA ligase I required for complementation.
Quantification of pulse-labeled replication products smaller than the
564-nucleotide marker in Fig. 4 confirmed that this population was not
altered in 46BR.1G1 extracts by supplementing them with either DNA
ligase III or T4 DNA ligase (data not shown). Human DNA ligase III
occurs as a heterodimer in vivo with XRCC1, a protein with
no known catalytic function (22). Incubation of recombinant human DNA
ligase III and XRCC1 in 46BR.1G1 extracts prior to pulse labeling did
not improve the ability of DNA ligase III to rescue the DNA replication
defect (data not shown).
Inhibition of in Vitro SV40 DNA Replication in Extracts of Normal Human Cells by Addition of DNA Ligase I or Its Amino-terminal Domain
The amino-terminal region of DNA ligase I is implicated in
interactions with other replication proteins, so it was of interest to
establish whether the addition of Hlig I-(2-263) in excess to
competent extracts could perturb DNA replication. In this instance, TAg-dependent dAMP incorporation into DNA during a 2-h SV40
DNA replication assay was estimated. Full-length DNA ligase I was found
to inhibit DNA replication in HeLa S3 extracts when added in
10-100-fold excess over the amount of DNA ligase I required to rescue
the replication defect in 46BR.1G1 extracts. Synthesis was completely
inhibited by the addition of full-length DNA ligase I to a final
protein concentration of 5 µM (Fig. 5).
The same inhibitory activity was seen with an excess of human DNA
ligase I lacking an amino-terminal His6 tag (data not
shown). When Hlig I-(2-263) and Hlig I-(262-919) were added
separately to the replication assay, the catalytic domain had no
significant effect on DNA replication even at 10 µM,
whereas Hlig I-(2-263) inhibited the reaction, although to a lesser
extent than the full-length protein; the amino-terminal domain alone
suppressed DNA synthesis by ~60% when added at 5 µM
(Fig. 5). These data indicate that the inhibition of DNA replication by
the addition of DNA ligase I to replication-competent normal cell
extracts is mediated via the amino-terminal domain of the protein.
Analysis of replication products on both neutral and denaturing agarose
gels showed no specific effect of DNA ligase I or its amino-terminal
domain on the joining of replication products in HeLa S3 extracts, but
rather suggested an overall suppression of DNA synthesis (data not
shown).
The SV40-transformed human fibroblast cell line 46BR.1G1 has a mutationally altered DNA ligase I protein with only ~5% of normal DNA joining activity. Studies using permeabilized, synchronized cells indicated that, while most newly synthesized Okazaki fragments were ligated at a normal rate in 46BR.1G1 cells, ~25% remained unligated for extended periods (13), in broad agreement with previous in vivo data (12, 23). In addition, 46BR.1G1 cells are hypersensitive to a wide range of DNA-damaging agents (24), suggesting a role for DNA ligase I in DNA excision repair process(es), and cellular hypersensitivity to simple alkylating agents could be normalized by transfection with the wild-type DNA ligase I cDNA (25). Here, we have added recombinant human proteins to soluble extracts supporting in vitro SV40 DNA replication and demonstrated the following: (i) complementation of aberrant DNA replication in 46BR.1G1 cell-free extracts by DNA ligase I, but not by other DNA ligases; (ii) a requirement for the amino-terminal non-catalytic region of DNA ligase I for efficient complementation; and (iii) inhibition of DNA replication in normal cell extracts by excess DNA ligase I or by the amino-terminal fragment of the protein.
During in vitro pulse-chase replication experiments, small DNA products, corresponding to the expected size of Okazaki fragments, were synthesized in both 46BR.1G1 and control MRC5 V1 extracts during the 20-s pulse. In MRC5 V1 extracts, these DNA fragments were rapidly converted into larger species, but in 46BR.1G1 extracts, unligated Okazaki fragments persisted during the chase period. Furthermore, in 46BR.1G1 extracts, but not in control extracts, pulse-labeled replication products appeared unstable, with only ~50% remaining after the 15-min chase. Titration of recombinant DNA ligase I protein into 46BR.1G1 extracts achieved complementation of both replication defects, and the full-length protein was ~10-fold more effective than the carboxyl-terminal catalytic domain alone. However, further addition of DNA ligase I protein was detrimental and, in 10-100-fold excess over the amount required to correct aberrant DNA replication in 46BR.1G1 extracts, caused inhibition of DNA replication in normal replication-competent extracts. A similar inhibitory activity was observed with the isolated amino-terminal domain. In contrast, the catalytic domain alone was not inhibitory even at high concentrations.
The identification of human DNA ligase I as a component of a 21 S protein complex that contains the activities required for in vitro SV40 DNA replication (26) indicates that DNA ligase I interacts with other components of the replication machinery. Adding excess DNA ligase I to cell extracts is likely to grossly perturb the stoichiometry governing such protein-protein interactions, and this may be sufficient to inhibit DNA replication. The inhibitory effect of the amino-terminal domain of DNA ligase I alone and the requirement for this domain to achieve efficient complementation of 46BR.1G1 extracts support the notion that the function of DNA ligase I during replication is mediated by specific binding of this amino-terminal non-catalytic domain to other replication protein(s). This concurs with in vivo studies showing that the first 115 amino acids of DNA ligase I are required for colocalization of the enzyme with bromodeoxyuridine at replication foci (10). As the amino-terminal fragment of DNA ligase I is not as potent an inhibitor of DNA replication as the full-length protein, folding of this region of the protein may be somewhat altered in the absence of the carboxyl-terminal catalytic domain. In addition, residues in the catalytic domain may be required to stabilize interactions of DNA ligase I with other replication factor(s) and could allow the catalytic domain alone to function weakly during replication in 46BR.1G1 extracts. Bacteriophage T4 DNA ligase and recombinant human DNA ligase III, which have no counterpart of the amino-terminal region of DNA ligase I, could not complement the replication defect in 46BR.1G1 extracts, in agreement with studies of SV40 DNA replication reconstituted with purified proteins (6); DNA ligase I was specifically required, DNA ligase III was unable to substitute, and T4 ligase substituted inefficiently. This is in marked contrast to both base excision repair and nucleotide excision repair in vitro, where no discrimination between different DNA ligases is observed in the joining step of the reconstituted pathways (15, 27).
The requirement for DNA ligase I during mammalian DNA replication suggests that it is an essential enzyme in vivo, and this has been directly demonstrated by gene knockout experiments in embryonic stem cells (9). However, in apparent conflict with these data, DNA ligase I null mouse embryos survived into midterm before dying of acute anemia (28). In this latter study, only the final four exons out of a total of 28 in the mouse DNA ligase I gene were deleted. The resulting truncated mRNA, which still encodes an intact amino-terminal domain and the active site of the enzyme, was reported to be unstable. However, low levels of this mRNA might generate a large fragment of DNA ligase I that, by analogy with a homologous fragment of bacteriophage T7 DNA ligase (29), could have enough residual activity to allow survival of null embryos, at least until very rapid cellular proliferation is required during early erythropoiesis.
Completion of lagging strand DNA synthesis requires RNase H1 to remove
the RNA primer, DNase IV/FEN-1 to cleave the final ribonucleotide
residue left by RNase H1, a DNA polymerase to fill the gap between
adjacent Okazaki fragments, and finally DNA ligase I to seal the nick
(4-6). In the absence of DNA ligation, the coordinated activities of
the DNase IV/FEN-1 nuclease and the DNA polymerase would catalyze nick
translation in the 5
3
direction at the junction of two Okazaki
fragments (30), and this may account for the observed instability of
unligated Okazaki fragments in 46BR.1G1 extracts. Moreover, PCNA has
been shown to interact directly with DNase IV/FEN-1 (31), and it has
been proposed that PCNA may remain bound to the DNA following Okazaki fragment synthesis to recruit DNase IV/FEN-1 and the gap-filling DNA
polymerase,
, or
(32). In consequence, the ends of unligated Okazaki fragments would not be generally accessible to nuclear proteins, and this model is consistent with the inability of human DNA
ligase III or T4 DNA ligase to function in lagging strand DNA
replication. Thus, the specificity of DNA ligase I is mediated by a
distinct amino-terminal domain, which probably allows access to an
otherwise protected nick via interaction with other components of the
replication machinery. DNA polymerase
or
, PCNA, and DNase
IV/FEN-1 are candidates for this role. Identification of the specific
protein partner(s) of DNA ligase I will further our understanding of
coordinated lagging strand DNA synthesis.
We thank Dr. Julian Blow for helpful discussions.