From the
DnaA protein loses the capacity to initiate chromosomal
replication when treated with a soluble cell extract. This inactivation
depends upon DNA and hydrolyzable ribonucleoside triphosphate. The
extract does not affect the activities of other replicative proteins or
the ability of DnaA to initiate replication of single-stranded DNA that
contains a DnaA-binding hairpin, indicating that the inhibitory effect
is specific for the action of DnaA at oriC. Gel filtration
experiments implicate a 150-kDa factor as being responsible. Mutant
DnaAcos protein, which causes overinitiation in vivo, is
insensitive to the inactivating factor, suggesting a requirement for
this negative control in vivo. We propose that a soluble
factor controls initiation through down-regulation of DnaA protein.
Initiation of genomic replication in Escherichia coli occurs at a unique origin ( oriC) on the chromosome and is
responsible for coupling replication to the cell cycle
(1, 2) . DnaA protein, the key initiation factor, binds
four 9-mer DnaA boxes in the minimal oriC origin (245 base
pairs). This complex promotes the opening of 13-mer AT-rich regions in
the minimal origin and directs the DnaC-mediated delivery of DnaB
helicase to the exposed single strands. The complementary strands are
subsequently synthesized by a replisome that includes DnaB, DnaG
primase, and DNA polymerase III holoenzyme.
The tight binding of ATP
and ADP to DnaA protein ( K
DnaA protein
is present at a level of >1000 molecules/cell
(6, 7) , although only
In vivo roles of DnaA protein include an
apparent regulation of the ``on-off switch'' of chromosomal
initiation. Overproduction of DnaA protein in vivo can shift
the timing of initiation to a point earlier in the cell cycle
(11) . Certain dnaA mutants can cause excessive
initiation
(7, 10, 12, 13, 14, 15, 16) .
The dnaAcos mutant is cold-sensitive (30 °C) for growth
and overinitiates the replication of its chromosome at the
nonpermissive temperature
(14) . Excessive initiation in
dnaAcos cells appears to be due to alterations in the
regulation of DnaA function rather than to an oversupply of the mutant
form of the protein
(7) . Whereas de novo protein
synthesis is absolutely necessary for new rounds of initiation in
wild-type dnaA cells, hyperinitiation in the dnaAcos mutant can occur without concomitant protein synthesis
(14) .
The specific activities of purified DnaAcos protein
and wild-type DnaA protein for catalyzing minichromosome ( oriC plasmid) replication in a crude in vitro system at 30
°C are very similar
(17) . However, there are significant
differences between DnaAcos protein and the wild-type form; DnaAcos
protein is unable to bind ATP and ADP, and as such, these nucleotides
are unable to influence the activity of the mutant protein. Strikingly,
DnaAcos protein also has the capacity to initiate replication for
longer periods in vitro; whereas initiation by wild-type DnaA
protein (0.40 pmol) ceases after 15 min in a crude replication system,
that by DnaAcos protein continues for at least 45 min
(17) .
These findings suggest the possibility that a considerable fraction of
wild-type DnaA protein in cells is inactivated, whereas DnaAcos protein
is not
(7) .
Here we present evidence that an activity in the
soluble fraction of a cell lysate is capable of inactivating wild-type
DnaA protein specifically for minichromosome replication. The
initiation potential of DnaAcos protein is not affected by exposure to
the soluble fraction. Thus, we propose that a soluble factor may be an
important regulator controlling initiation frequency. The insensitivity
of DnaAcos protein to the negative factor coincides with the
nonattenuated activity in vitro and the overinitiation trait
in vivo.
The oriC plasmid (M13mpRE85) was
included with the cell extract for this inactivation. Inasmuch as
adequate superhelicity is required for oriC plasmids to serve
as templates for replication in vitro (27) , nicking of
template DNA by endonucleases in the protein extract might be
responsible for the observed inefficient replication. However, agarose
gel electrophoresis did not reveal any significant changes in the
amount or form of template DNA present in the mixture prior to
replication (Fig. 1 C). Thus, such changes are not the cause of
the loss of replication activity (also see below).
DnaA protein-dependent replication of ssDNA
(template termed M13 A-site) was examined to see whether or not the
inactivation of DnaA protein is specific for initiation at
oriC. DnaA protein was incubated with the cell extract;
portions of the mixture were added to replication reactions; and DNA
synthesis initiated at oriC or the hairpin on M13 A-site ssDNA
was measured. While DnaA protein was unable to initiate replication at
oriC, its action in this ssDNA replication system was
indistinguishable from that of DnaA protein that had not been exposed
to the cell extract (Fig. 4). This finding supports the observation
that DnaA protein is not globally altered (Fig. 1 B) and
indicates that a factor in the cell extract inactivates some function
of DnaA protein that is required for initiation at oriC but
that is dispensable for initiation of ssDNA replication.
Chromosomal replication is regulated at the initiation stage,
most likely through a balance of positive- and negative-acting factors.
Here we have indicated that a soluble cellular factor inactivates DnaA
protein for initiating replication at oriC in vitro and
propose that such a negative factor plays an important role in
controlling initiation in vivo. A mutant form of DnaA protein,
DnaAcos, which overinitiates replication at oriC, is not
affected by exposure to the factor; the inability to respond to such a
negative-acting factor may be deeply involved in the overinitiation
observed for dnaAcos mutants. Unlike wild-type DnaA protein
activity, DnaAcos protein activity for initiation in vitro is
thermosensitive, and its specific activity at 42 °C is only 25% of
that at 30 °C
(17) . Since the dnaAcos mutant can
grow normally at 42 °C, these facts also support the importance of
down-regulation of initiation activity.
Considerable portions of
DnaA and DnaAcos proteins are found as aggregates during their
purification from overproducing cells
(6, 17, 18) . Aggregates of wild-type DnaA
protein include phospholipids with a composition similar to that of
cell membranes
(23) . The initiation activity of the aggregate
form is about half of that of monomeric DnaA protein in a crude
replication system and is completely absent in a system reconstituted
with purified proteins
(23) . The inactivated DnaA protein
observed here, however, differs from the aggregated form; constant
velocity centrifugation through sucrose indicated that a large majority
(>80%) of DnaA protein was monomeric following treatment with the
crude extract either in the presence or absence of
Genetic analyses and reconstitution
studies with purified components have identified >10 different
proteins that are necessary for replication from oriC (1) . Among these numerous participants, DnaA protein seems
to be the only factor that is inactivated by the soluble crude extract
(Fig. 5). This finding supports the hypothesis that the
inactivating factor has a specific role in the control of chromosome
initiation during cell growth.
We found that the inactivation of
DnaA protein is specific for initiation at oriC; initiation of
A-site ssDNA replication is not affected (Fig. 4). This finding
suggests that the DNA-binding and DnaB helicase-loading activities of
DnaA protein are not affected. Perhaps DnaA protein's additional
function of strand separation of the AT-rich region of oriC is
what is lost.
Although the slow conversion of ATP-DnaA protein to the ADP form can
occur independently of other cellular components
(3) , the
inactivating factor may stimulate the feeble intrinsic ATPase. In the
regulation of eukaryotic cell growth, a similar example is seen in the
oncogenic Ras GTPase; the very weak GTPase of Ras protein is stimulated
by interaction with GTPase-activating protein, and the GTP-bound form
active for signaling cell proliferation is rendered to the GDP-bound
inactive form
(30) . DnaA protein inactivated by the 150-kDa
factor appears to resemble the ADP form that is inert for initiation at
oriC (3) , but is active for initiation of A-site ssDNA
(22) .
A protein termed IciA
( inhibitor of chromosomal initiation) was
previously shown to prevent the strand opening at oriC and to
act as a specific inhibitor of initiation in vitro (31) . The action of the factor we describe here differs
from that of IciA protein in that (i) IciA protein must be bound to the
13-mer region of oriC to inhibit the initiation activity of
DnaA protein, while the proposed inactivating factor can restrain the
action of DnaA protein in the absence of oriC ();
(ii) IciA protein is a homodimer of 33-kDa subunits, as opposed to the
apparent native molecular mass of
A rapidly growing bacterium (doubling time of
<60 min) must initiate the next round of chromosomal replication
before ongoing replication has been completed and the cell divides and
thus will bear multiple copies of oriC (32) .
Initiation of the multiple origins within a cell is synchronous,
occurring at the same time during the cell cycle. Modification of
adenine residues in the sequence GATC by DNA-adenine methylase (the
dam gene product)
(33) is essential for this synchrony
(2) ; dam
The recently identified seqA gene encodes a protein that appears to negatively modulate
replication by sequestration
(39) . seqA-deficient
cells are viable, but cannot sequester oriC DNA or synchronize
initiations. The action of SeqA protein in controlling initiation is
independent of the proposed negative regulation through the
inactivation of DnaA protein. An extract prepared from seqA null cells had the same specific activity for inactivating DnaA
protein as wild-type cells and did not affect DnaAcos activity (data
not shown). Thus, SeqA protein and the DnaA-inactivating factor appear
to act independently to affect initiation at oriC.
Sequestration of oriC to the membrane is transient during
the cell cycle, with the duration being much shorter than the time
between initiations
(38) . As such, it is likely that an as yet
unidentified factor functions to determine the occurrence of initiation
during the cell cycle. The DnaA-inactivating factor may play a role in
this regulation. DnaAcos protein is insensitive to the factor and thus
may retain an initiation-competent state for an extended period and
cause repeated initiations. A system for inactivating DnaA protein may
be necessary for controlling the on-off switch of initiation during the
cell cycle. Purification of the inactivating factor is in progress.
Wild-type DnaA or DnaAcos protein (3.8 pmol) was
added to a standard first-stage reaction (25 µl) that lacked or
included DNA (1.0 µg for double-stranded DNA; 0.5 µg for
ssDNA). Portions (5 µl) of the mixtures, either before or after an
incubation period (40 min, 30 °C), were added to an in vitro DNA synthesis complementation reaction (20 min, 30°C) that
contained M13mpRE85 as template DNA (see ``Experimental
Procedures''). When M13mpRE85 was present in the first-stage
reaction, template DNA was not added to the replication reaction. DnaA
protein activity remaining after the first-stage incubation (residual
activity) was compared with that obtained before and is indicated in
percent (in each case, an activity of 100% corresponds to 400-600
pmol of DNA synthesized).
DnaA protein was incubated with
ATP (5 µ
M; 15 min, 0 °C) to form the nucleotide-bound
form. The resulting ATP-DnaA protein (3.8 pmol) was added to a standard
first-stage reaction (25 µl) that contained the indicated
nucleotides. Portions (5 µl) of the mixtures, either before or
after an incubation period (40 min, 30 °C), were added to an in
vitro DNA synthesis complementation reaction (20 min, 30 °C;
see ``Experimental Procedures''). DnaA protein activity
remaining after the first-stage period (residual activity) was compared
with that obtained before and is indicated in percent (in each case, an
activity of 100% corresponds to 300-400 pmol of DNA synthesized).
We are grateful to Drs. Nicholas E. Dixon and Hisao
Masai for the gift of M13 A-site ssDNA. We also thank Drs. Kazuhisa
Sekimizu, Hisaji Maki, and Erik Boye for suggestions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 0.03
and 0.1 µ
M, respectively) has been shown to have a
profound influence on the protein's activity for initiation
in vitro (3, 4, 5) . The ATP-bound form
of DnaA protein is active for initiating replication; the ADP form,
while capable of binding oriC, is inefficient for catalyzing
strand separation and thus feeble for initiation. However, the cellular
presence of the nucleotide-bound form is still unknown.
20 molecules of the protein
seem to be involved in the oriC complex
(8, 9) . The cellular amount and synthesis of DnaA
protein do not oscillate during the cell division cycle, and the
protein is stable for at least 90 min in growing cells
(10) .
Since initiation occurs properly in a strain in which the dnaA gene is expressed constitutively using the lac promoter
(11) , modulation of DnaA protein activity may be responsible
for regulating initiation rather than cyclic expression of the dnaA gene.
Materials
Sources were as follows:
deoxyribonucleoside triphosphates and Sephacryl S-300 HR, Pharmacia
Biotech Inc.; ribonucleoside di- and triphosphates, HEPES,
phosphocreatine, and rifampicin, Sigma;
[-
P]TTP (800 Ci/mmol), Amersham Corp.;
creatine kinase and ATP
S,
(
)
Boehringer
Mannheim;
X174 RF I DNA, Life Technologies, Inc.; and
DEAE-cellulose (DE52), Whatman.
In Vitro DNA Synthesis Complementation
Reaction
Standard reactions (25 µl) contained 40 m
M HEPES/KOH (pH 7.6 at 1
M); 2 m
M ATP; 0.5 m
M each CTP, GTP, and UTP; 100 µ
M each dATP, dCTP, dGTP,
and [-
P]TTP (70-130 cpm/pmol); 11
m
M magnesium acetate; 7% (w/v) polyvinyl alcohol
( M
30,000-70,000); 40 m
M phosphocreatine; 100 µg/ml creatine kinase; crude fraction II
cell extract
(18) (200-250 µg of protein) prepared
from WM433 ( dnaA204) cells
(15) ; and M13mpRE85
(19) oriC plasmid template DNA (600 pmol as nucleotide;
38 fmol as circle). The reactions were complemented with purified
monomeric DnaA (52 kDa; 0.96
10
units/mg) or
DnaAcos (0.84
10
units/mg) protein and incubated
for 20 min at 30 °C. Nucleotides incorporated into acid-insoluble
material were trapped on GF/C filters and measured by liquid
scintillation counting.
Inactivation of DnaA Protein (First-stage
Reaction)
Reactions were assembled at 0 °C and incubated at
30 °C for the indicated times. Typically, reactions (25 µl)
contained 40 m
M HEPES/KOH (pH 7.6 at 1
M), 2 m
M ATP, 11 m
M magnesium acetate, 7% (w/v) polyvinyl alcohol
( M30,000-70,000), 40 m
M phosphocreatine, 100 µg/ml creatine kinase; 1.0 µg of
M13mpRE85 or
X174 RF I DNA (3.0 nmol as nucleotide), 3.8 pmol of
wild-type DnaA or DnaAcos protein, and 200 µg (unless indicated
otherwise) of a WM433 extract (fraction II noted above). Portions (5
µl) of the mixtures were added to in vitro DNA synthesis
reactions (second-stage reaction) to measure DnaA protein activity
remaining after the inactivation procedure (``residual
activity''). One unit of inactivation corresponds to a 1-unit
decrease in the specific activity of DnaA protein/min at 30 °C.
Formation of ATP-DnaA Protein
DnaA protein (11.5
pmol) was added to mixtures (15 µl) containing 50 m
M HEPES/KOH (pH 7.6 at 1
M), 2.5 m
M magnesium
acetate, 0.3 m
M EDTA, 20% (v/v) glycerol, 0.007% Triton X-100,
7 m
M dithiothreitol, and 1 µ
M ATP and incubated
for 15 min at 0 °C
(3, 20) . For nucleotide-free
DnaA protein, the same conditions were used, except that ATP was
omitted.
ABC Primosome System for ssDNA
Replication
Reaction conditions were the same as those described
for replication of X174 ssDNA
(21) , except for the
addition of rifampicin (40 µg/ml), phosphocreatine (50
m
M), and creatine kinase (100 µg/ml). Each reaction (20
µl) included WM433 fraction II crude extract (100 µg of
protein) and M13 A-site ssDNA (300 pmol as nucleotide; 41 fmol as
circle)
(22) as template. DnaA protein (provided as a component
of the first-stage reaction) was added, and the samples were incubated
at 30 °C for 10 min. Nucleotides incorporated into acid-insoluble
material were trapped on GF/C filters and measured by liquid
scintillation counting.
Other Methods
Immunoblot analysis
(7) ,
density centrifugation
(23) , and agarose (7%) gel
electrophoresis
(24) were performed as described. Commercially
obtained X174 RF I DNA was further purified by treatment in
ammonium acetate (2.6
M) and ethidium bromide (0.4 mg/ml),
extracted twice with phenol/chloroform (1:1), and precipitated in
ethanol as described
(25) . Protein concentrations were measured
with the Bradford method
(26) using bovine serum albumin as a
standard.
DnaA Protein Is Inactivated in a Soluble Cell Extract;
DnaAcos Protein Is Not
Unlike wild-type DnaA protein, DnaAcos
protein is capable of catalyzing multiple rounds of replication in
vivo without de novo protein synthesis
(14) . In a
crude in vitro replication system, DnaAcos protein can
initiate replication of oriC plasmids for a longer time than
the wild-type form
(17) . Since an unknown secondary factor may
modulate DnaA protein activity, we have examined whether the activities
of wild-type DnaA and DnaAcos proteins are altered when incubated with
a crude cell extract (Fig. 1).
Figure 1:
Wild-type DnaA protein, but not DnaAcos
protein, loses activity following exposure to a soluble cell extract.
DnaA and DnaAcos proteins (7.7 pmol) were added to standard first-stage
reactions (50 µl) that contained M13mpRE85 (2.0 µg) and were
incubated at 30 °C (see ``Experimental Procedures'').
A, at the indicated times, 5-µl portions were transferred
to in vitro DNA synthesis complementation reactions (25
µl; 20 min, 30 °C) (see ``Experimental Procedures'').
DnaA protein activities remaining after the first-stage periods
( Residual activity) were compared with those obtained
without the first-stage incubation (time = 0 min). Activities of
100% correspond to 464 and 390 pmol of DNA synthesized for wild-type
DnaA protein (DnaA) and DnaAcos protein, respectively.
B, at the indicated times, 5-µl portions of the
first-stage reaction including wild-type DnaA protein were mixed with
trichloroacetic acid (100 µl of a 5% solution) and kept on ice for
at least 20 min. Precipitated proteins were dissolved in
SDS-polyacrylamide gel electrophoresis sample buffer, and levels of
DnaA protein were determined by immunoblotting with anti-DnaA protein
antiserum (7). The arrow identifies the band for DnaA protein.
The background control, which included all components except for DnaA
protein, was similarly treated. C, at indicated times,
5-µl portions were treated with SDS (150 µl of a 0.5%
solution), and M13mpRE85 DNA (7.9 kilobases) was extracted with phenol
and precipitated in ethanol (27). Recovered DNA was analyzed by agarose
(7%) gel electrophoresis. DNA was visualized with ethidium bromide (0.5
µg/ml). The sizes of molecular size markers
( HindIII-digested
DNA) are indicated in kilobases
( kb). F I, F II, and F III indicate the migration of supercoiled, nicked
circular, and linearized forms, respectively, of M13mpRE85
DNA.
While DnaAcos protein remained
active after exposure to a soluble protein extract, the activity of
wild-type DnaA protein decreased to background levels
(Fig. 1 A). Immunoblot analysis with anti-DnaA serum
detected no significant decrease in either the level of DnaA protein or
its apparent molecular mass during treatment with the protein extract
(Fig. 1 B). Thus, the inactivation of DnaA protein does
not seem to be due to overall proteolytic degradation, a result
consistent with the stability of DnaA protein in vivo (10) .
Inactivation Depends on a Soluble Cell Extract
The
inactivation we observed here (Fig. 1 A) required the
inclusion of a cell extract; mixtures in which the extract was replaced
with bovine serum albumin in buffer (Fig. 2 A) or
HO (data not shown) were unable to inactivate DnaA protein.
Furthermore, DnaA protein must be exposed to the extract for its
inactivation to occur. Replication did not decrease when the extract
was first incubated without DnaA protein and then mixed with DnaA
protein and added to replication reactions (Fig. 2 B).
The protein extract seems to act directly upon DnaA protein during the
first incubation and does not generate an inhibitor for the subsequent
replication reaction.
Figure 2:
The
soluble extract is responsible for the inactivation of DnaA protein.
A, DnaA protein (3.8 pmol) was added to first-stage reactions
(50 µl) that contained M13mpRE85 (2.0 µg) and either a soluble
extract () or bovine serum albumin (400 µg;
) and was
incubated at 30 °C. At the indicated times, 5-µl portions of
the mixtures were transferred to in vitro DNA synthesis
complementation reactions (25 µl; 20 min, 30 °C). DnaA protein
activities remaining after the first-stage incubation ( Residual activity) were compared with those obtained without
inactivation (time = 0 min). Activities of 100% correspond to
249 and 220 pmol of DNA synthesized for mixtures containing extract and
bovine serum albumin, respectively. B, the first-stage
reactions (25 µl) with (
) and without (
) DnaA protein
(3.8 pmol) were incubated at 30 °C. At the indicated times,
5-µl portions were transferred to in vitro DNA synthesis
complementation reactions (25 µl; 20 min, 30 °C); DnaA protein
(0.77 pmol) was added to samples that lacked DnaA protein during the
first stage. Activities of 100% correspond to 411 and 452 pmol of DNA
synthesized for inactivation reactions with and without DnaA protein,
respectively. C, DnaA protein (7.7 pmol) was added to
first-stage reactions (50 µl) containing varied amounts of a
soluble extract and was incubated at 30 °C for 40 min. Samples (5
µl, with the indicated amounts of extract protein) were transferred
to in vitro DNA synthesis complementation reactions (25
µl; 20 min, 30 °C). DnaA protein activities remaining after the
first stage (residual activity) were compared with those obtained
without extract. A residual activity of 100% corresponds to 515 pmol of
DNA synthesized.
The extent of inactivation was proportional to
the amount of extract added (Fig. 2 C), further
suggesting that a factor(s) in the extract is responsible for the
inactivation of DnaA protein. The inactivation activity of the extract
is heat-labile in that heating the extract at 55 °C for 5 min
prevented the extract from being able to act upon DnaA protein (data
not shown).
Inactivation of DnaA Protein Is DNA-dependent
The
inclusion of DNA during the treatment of DnaA protein with the cell
extract is necessary for inactivation to occur. When DnaA protein was
incubated with the extract (40 min) in the presence of oriC template DNA (M13mpRE85) and then the mixture was added to a
replication reaction, 80% of the replication activity was lost
(Fig. 1 A and ). In contrast, significant
replication was observed when DnaA protein was incubated with the
extract (40 min) in the absence of DNA and then assayed in a
replication reaction in which template DNA was provided ().
However, the need for specific DNA sequences, such as a DnaA box, was
not found in that M13mp19 RF I, X174 RF I, and
X174 ssDNA
supported the inactivation of DnaA protein (). The ability
of nontemplate DNA to promote the inactivation process confirms the
finding that the loss of replication activity is not caused by changes
in template DNA (Fig. 1 C).
Inactivation of DnaA Protein Is ATP-dependent
ATP
was essential for the inactivation of DnaA protein (Fig. 3 A),
with maximal inactivation occurring at concentrations of 100 µ
M or higher (Fig. 3 B). The requirement for ATP was
similar when either the nucleotide-free or ATP-bound form of DnaA
protein was used (Fig. 3, A and B). Thus, the
exogenous ATP appeared not to be necessary for the formation of the ATP
form of DnaA protein and may be needed for the inactivating factor.
Figure 3:
ATP requirement for the inactivation of
DnaA protein. A, nucleotide-free DnaA protein (7.7 pmol;
and
) or ATP-bound DnaA protein (7.7 pmol;
and
)
(see ``Experimental Procedures'') was added to first-stage
reactions (25 µl) with (
and
) and without (
and
) ATP (2 m
M) and was incubated at 30 °C. At the
indicated times, 5-µl portions were transferred to in vitro DNA synthesis complementation reactions. Residual DnaA protein
activities were calculated as described in the legend to Fig. 1. A
residual activity of 100% corresponds to 350-450 pmol of DNA
synthesized. B, nucleotide-free DnaA protein (0.77 pmol;
) and ATP-bound DnaA protein (0.77 pmol;
) were added to
first-stage reactions (5 µl; 30 min, 30 °C) with the indicated
levels of ATP. DnaA protein activities were measured in the
complementation reaction (25 µl; 20 min. 30 °C), and the
residual activities were calculated. Residual activities of 100% for
nucleotide-free and ATP-bound forms correspond to 464 and 412 pmol of
DNA synthesized, respectively.
Compounds related to ATP were used to examine the nucleotide
specificity for DnaA protein inactivation. To prevent DnaA protein from
forming complexes with the ATP analogs, the ATP-bound form of DnaA
protein was used. Clearly, CTP, GTP, and UTP at 0.5 m
M sustained inactivation of ATP-DnaA protein, whereas ADP and the
nonhydrolyzable analog ATPS could not ().
DnaA Protein Is Inactivated Specifically for Initiation
at oriC
DnaA protein participates in the assembly of primosomes
at sites other than oriC (22, 28, 29) .
The ABC primosome system requires DnaA, DnaB, and DnaC proteins; DnaG
primase; and E. coli single strand binding protein-coated
ssDNA that contains a hairpin structure with a single DnaA box
(22) . DnaA protein binds to its recognition sequence and, with
the aid of DnaC protein, loads the DnaB helicase onto the template.
DnaG primase then forms a complex with DnaB protein and synthesizes
primer RNA on ssDNA.
Other Replication Components in the Cell Extract Are
Unaffected by Prolonged Incubation
The rate of DNA synthesis in
a crude cell extract complemented with DnaA protein (0.19 pmol)
decreased after 10 min (Fig. 5). With the addition of fresh
DnaA protein, replication was revived and continued for an additional
20 min (Fig. 5). The extent of the additional DNA replication was
proportional to the amount of DnaA protein added. This finding
indicates that DnaA protein in a crude cell extract loses much of its
initiation capacity, while the other factors needed for DNA synthesis
retain their activities.
Figure 5:
DNA synthesis arrested in a crude extract
is reinstated with additional DnaA protein. DNA synthesis
complementation reactions (25 µl) containing DnaA protein (0.19
pmol) were incubated (30 °C) for the indicated times (see
``Experimental Procedures''). Additional DnaA protein was
added to some reactions ( and
) after 20
min.
Fractionation of the Cell Extract by Gel
Filtration
The crude cell extract (12 mg, 3.9 10
units) was fractionated through a gel filtration column to
determine if the DnaA protein-inactivating factor behaved as a single
entity. Column fractions were incubated with
X174 RF I DNA, ATP,
and DnaA protein, and then replication activities were measured. A peak
decrease in DnaA protein activity was observed for fraction 33, which
corresponds to an apparent molecular mass of
150 kDa
(Fig. 6). The yield of the inactivating factor activity in a pool
of fractions 31-39 was 62% of the loaded activity. The loss of
DnaA protein activity required incubation of DnaA protein with the
column fractions (Fig. 6), and as expected, the inclusion of ATP
and DNA in the inactivating step was necessary (data not shown).
Figure 6:
Size-exclusion chromatography of the
soluble extract. The soluble extract (fraction II; see
``Experimental Procedures'') was applied to a Sephacryl
HR-300 column (1.3 28 cm, 37 ml) equilibrated with 25 m
M HEPES/KOH (pH 7.6 at 1
M), 0.1 m
M EDTA, 2 m
M dithiothreitol, 50 m
M KCl, and 20% sucrose. Proteins were
eluted with the same buffer (flow rate of 0.3 ml/min), and fractions
(0.33 ml) were collected. Portions (3.9 µl) of column fractions
were added to first-stage reactions (11 µl; containing 600 ng of
X174 RF I DNA) and incubated with DnaA protein (2.3 pmol).
Portions (5 µl) of the mixtures before (
) and after (
)
incubation (30 min, 30 °C) were assayed for replication activity in
in vitro DNA synthesis complementation reactions as described
in the legend to Fig. 1. Markers are catalase ( cata; 232 kDa),
bovine serum albumin ( BSA; 68 kDa), and cytochrome c ( cyto; 12 kDa).
Fractionation Profile through a DEAE-cellulose
Column
While the inactivating factor failed to bind to several
chromatographic resins (data not shown), it had affinity for
DEAE-cellulose (DE52). Fractions eluted from a DE52 column by a linear
gradient of sodium chloride were assayed for the inactivation of DnaA
protein (Fig. 7); one major peak of DnaA-inactivating activity
was observed. The yield of activity in the pool of fractions
27-43 was 54%. This pooled fraction had a 4.5-fold increase in
specific activity over fraction II and required ATP and X174 RF I
DNA for inactivation (data not shown).
Figure 7:
Anion-exchange chromatography of the
soluble extract. The soluble extract was prepared as described under
``Experimental Procedures,'' except that proteins were
precipitated with 0.235 mg/ml ammonium sulfate. Fraction II proteins
(24 mg, 4.1 10
units) were loaded onto a DE52
column (1.3
2.3 cm, 3 ml) equilibrated with 25 m
M BisTris/HCl (pH 6.1), 0.1 m
M EDTA, 2 m
M dithiothreitol, 15% glycerol, and 100 m
M sodium chloride.
Activity was eluted in the same buffer by a linear gradient (30 ml)
from 100 to 250 m
M sodium chloride. Portions (5 µl) of
column fractions were added to first-stage reactions (10 µl;
containing 600 ng of
X174 RF I DNA) and incubated with DnaA
protein (1.0 pmol). Portions (5 µl) of these reactions after
incubation (40 min, 30 °C) were assayed for replication activity in
in vitro DNA synthesis complementation
reactions.
X174 RF I DNA
and ATP (data not shown).
Figure 4:
Replication activity of DnaA protein
treated with a soluble extract for synthesis of M13 A-site ssDNA and
minichromosomes. DnaA protein (8.7 pmol) was added to a first-stage
reaction (75 µl). Before ( and
) and after (
and
) a period of incubation (40 min, 30 °C), the indicated
volumes of the reaction were transferred into complementation reactions
(20 min, 30 °C) for minichromosome replication (600 pmol (as
nucleotide) of M13mpRE85 template DNA;
and
) and for the
ABC primosome system for single-stranded DNA synthesis (300 pmol (as
nucleotide) of M13 A-site ssDNA template DNA;
and
; see
``Experimental Procedures'').
A possible molecular mechanism for the inactivation of
DnaA protein may include enhanced hydrolysis of the tightly bound ATP.
Wild-type DnaA protein has a weak intrinsic ATPase activity; tightly
bound ATP is very slowly hydrolyzed to ADP at 37 °C in the presence
of DNA, thereby generating replicatively inefficient ADP-DnaA protein
(3) . The requirement of sequence-independent DNA for the
hydrolysis of the bound nucleotide
(3) is similar to that
described here for the inactivation of DnaA protein ().
(
)
The requisite hydrolysis of a
ribonucleotide to inactivate DnaA protein () suggests that
the inactivation process may either be energy-consuming or involve a
phosphorylation event. However, phosphorylated forms of DnaA protein
have not been detected (data not shown).
150 kDa observed for the DnaA
protein-inactivating factor (Fig. 6); and (iii) there is an
absolute requirement for a nucleoside triphosphate for the inactivation
of DnaA protein ( Fig. 3and Table II). None is required for IciA
protein to function.
cells with either point or
null mutations are viable
(34, 35) , but exhibit an
asynchronous phenotype
(36) . In wild-type cells,
semiconservative replication of fully methylated origins produces
hemimethylated oriC DNA that binds to the cell membrane
(37) . The hemimethylated state of oriC seems to be
maintained for 10 min
(38) ; and then Dam methylase modifies the
GATC sites in the oriC region, and the resultant fully
methylated DNA is released from the membrane. This process, termed
sequestration, plays a role in establishing the synchrony of
initiations of multiple chromosomes and preventing secondary initiation
at newly replicated origins.
Table: DNA requirements for the inactivation of
DnaA protein
Table: Nucleotide requirements for the
inactivation of DnaA protein
S, adenosine 5`- O-(thiotriphosphate); RF, replicative
form; ssDNA, single-stranded DNA; BisTris,
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.