From the Department of Microbiology, Duke University
Medical Center, Durham, North Carolina 27710, the
§ Department of Biological Chemistry, Structural Biology
Group, Chiron Corporation, Emeryville, California 94608, the
¶ Department of Human and Molecular Genetics, Baylor College of
Medicine, Houston, Texas 77030, the
Centro de Biologia Molecular
Severo Ochoa, Universidad Autonoma, Canto Blanco, 28049 Madrid, Spain,
and the ** Department of Genetics, Groningen Biomolecular Sciences and
Biotechnology Institute, Haren, The Netherlands
Received for publication, December 4, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have delineated the amino acid to nucleotide
contacts made by two interacting dimers of the replication terminator
protein (RTP) of Bacillus subtilis with a novel
naturally occurring bipolar replication terminus by converting RTP to a
site-directed chemical nuclease and mapping its cleavage sites on the
terminus. The data show a relatively symmetrical arrangement of the
amino acid to base contacts, and a comparison of the bipolar contacts
with that of a normal unipolar terminus suggests that the DNA-protein
contacts play an important determinative role in generating polarity
from structurally symmetrical RTP dimers. The amino acid to nucleotide contacts provided distance constraints that enabled us to build a
three-dimensional model of the protein-DNA complex. The model is
consistent with features of the bipolar Ter·RTP complex
derived from mutational and cross-linking data. The bipolar terminus
arrested Escherichia coli DNA replication and DnaB helicase
and T7 RNA polymerase in vitro in both orientations. RTP
arrested the unwinding of duplex DNA on the bipolar Ter DNA
substrate regardless of the length of the duplex DNA. The latter
result suggested further that the terminus arrested authentic DNA
unwinding by the helicase rather than just translocation of helicase on DNA.
In many prokaryotic and some eukaryotic replicons, replication
forks initiated at specific replication origins and moving bi-directionally are not terminated randomly but in regions delimited by polar replication termini
(Ter).1 The
Ter sites are usually short sequences that specifically bind to a replication terminator protein (RTP) and arrest fork moving in
only one direction with respect to the origin but not the other. The
Ter sites are usually located in two clusters of opposite polarity in such a way that the forks moving clockwise on a circular chromosome pass through the first cluster (that has the nonarresting polarity) and are arrested at the termini of the second cluster, which
has blocking polarity. The same is true for forks moving in a
counterclockwise direction (1, 2).
Each Ter site of Bacillus subtilis binds to two
interacting dimers of RTP to arrest forks in a polar mode (3, 4). Thus, there are two interesting mechanistic questions to be addressed in this
context. First, how is the polarity of fork arrest generated, considering the fact that the dimeric RTP has a symmetrical structure (5). Second, what is the molecular mechanism of fork arrest (see
reviews in Refs. 2 and 6). In this study we have endeavored to address
the first question.
We have approached the question by determining the amino acid to
nucleotide contacts of RTP bound to the novel naturally occurring bipolar terminus of the plasmid pLS20 (7) and comparing and contrasting
the results with similar contacts derived from a normal unipolar
Ter site. We have converted RTP to a site-directed chemical nuclease by coupling it to an organic Fe-EDTA conjugate at certain rationally selected critical amino acid residues that are known to be
involved in contacting Ter DNA (8-10) and have determined the site-directed cleavage maps of the bipolar replication terminus. Furthermore, using the crystal structure of RTP (5) and the cleavage
maps mentioned above, we have constructed a model of the two dimers of
RTP bound to the bipolar Ter. The model revealed a
relatively symmetrical amino acid to nucleotide contact pattern that
presumably contributed to nearly equal binding affinity of both
subsites of the bipolar Ter to RTP. The results suggest that the pattern of protein-DNA interaction at the Ter sites is
probably one of the parameters that generate polarity (or the lack of
it) at the Ter sites.
In the second part of this study, we have investigated the biochemical
properties of the bipolar Ter with the goal of mapping the
points of arrest of the helicase and T7 RNA polymerase at each end of
the Ter site and have determined the minimal effective sequence necessary to arrest the helicase. We have determined an
"activity footprint" of the contrahelicase activity of RTP on the
bipolar Ter that showed that RTP was able to arrest
helicase-catalyzed unwinding of double stranded DNA in a
length-independent fashion over a range of a less than a 100 bp to over
1500 bp. This result is consistent with the notion that RTP arrested
authentic DNA unwinding and not just helicase translocation. Thus, the
data presented provide not only some insight into the molecular basis of the origin of polarity but also the characteristics of the bipolar
terminus in vitro.
Recombinant DNA Constructs--
The pUC18-IRI (BS3) and
pUC18-IRI (BS3) Rev. plasmids were constructed by transferring an
XbaI-HindIII fragment that contained the IRI
(unipolar Ter) from the pET22b-BS3 and pET22b-BS3 Rev. plasmids (11) into the pUC18 vector. The pUC18/19-Bipolar plasmids were
constructed by cloning a 46-bp EcoRI-HindIII
fragment containing the bipolar Ter site into the pUC18/19
vectors. The plasmids pET22b-IRI, (pET22b-BS3) and pET22b-IRI Rev.
(pET22b-BS3 Rev) contained a unipolar Ter site in opposite
orientations. The pET22b-Bipolar and pET22b-Bipolar Rev. plasmids were
constructed by cloning an NdeI-HindIII and an
NdeI-EcoRI fragment from pUC18-Bipolar and pUC19-Bipolar plasmids, respectively, in the pET22b vector.
M13mp19-Bipolar and M13mp18-Bipolar clones were made by cloning a 46-bp
DNA fragment containing the bipolar terminus into the M13mp18 and
M13mp19 vectors, respectively.
Conversion of RTP to a Chemical Nuclease and Cleavage Maps of
RTP-Bipolar Ter Complex and Generation of a Three-dimensional Model of
the Complex--
RTP contains a single and apparently
solvent-inaccessible Cys at position 110 in the dimerization
To maintain the integrity of the planar nucleotide bases, a planarity
term was included to prevent buckling of the bases. It should be noted
that the models yielded by the simulation are qualitative in nature and
do not approach the rigor of an all-atom molecular dynamics simulation.
For example, whereas the simulations will provide information on how
each dimer binds its cognate DNA, it is impossible to exactly define
specific side chain-base contacts from the resulting model.
In Vitro DNA Replication--
Preparation of cell extracts and
reaction conditions were as published (12) with some modifications.
Transcription Assays--
This was carried out as described (11,
13).
Electrophoretic Mobility Shift Assays, Helicase Assays, Helicase
Ladder Assay, and Methylation Protection Assays--
These have been
described before (14, 15).
Methylation Protection Analysis of RTP-Bipolar Ter
Complex--
Our first goal was to derive a picture of the arrangement
of two interacting dimers of RTP at the bipolar Ter. A
priori, a comparison of the bipolar Ter with a unipolar
Ter (IRI) sequence showed significant homology at the core
sequence and very little at the auxiliary sequence (Fig.
1A). A closer look suggested
that the auxiliary site-equivalent of the bipolar Ter has
significant sequence homology with core sequence, and thus the bipolar
Ter may have two core sequences arranged in a head-to-head
manner (Ref. 7, see Fig. 1A). To determine some of the key
base to amino acid contacts between the two RTP dimers and the bipolar Ter DNA, the protein was complexed with 5'end-labeled DNA
and treated with dimethyl sulfate as described (14), and the methylated bases were mapped after depurination and
An examination of the guanine contacts of the bipolar Ter
site showed a symmetrical pattern as contrasted with that of the normal
unipolar Ter (IR1) site that showed more G contacts at its
core sequence than the auxiliary site (Fig. 1B). This result prompted us to investigate in more detail the amino acid to nucleotide contacts between RTP and the bipolar Ter site as described below.
Computational Modeling of the RTP-Bipolar Terminus DNA
Complex--
Ideally, one needs to solve the x-ray crystal structure
of the RTP·Ter complex to develop a detailed picture of
how the protein interacts with DNA. However, the co-crystals of bipolar
Ter·RTP complex that have been generated up to this time
diffract to ~6 Å. Although further improvement in resolution and the
eventual determination of the structure looks
promising,2 we decided to
approach the problem in another way. We converted RTP to a
site-directed chemical nuclease and used it to generate an amino acid
to nucleotide cleavage map of the RTP-bipolar Ter complex.
The structure of (S)-(2-pyridylthio) cysteaminyl-EDTA (EPD),
the exchange reaction with cysteine-substituted RTP, and the expected
pattern of cleavage when the iron atom contacts the major as contrasted
with the minor groove of the DNA are shown in Fig.
2. The cleavage data were used along with
computational methods described below, to construct a three-dimensional
model of the RTP-bipolar Ter complex.
Such models have served useful purposes for studies of DNA-protein
interaction in solution and provide a useful adjunct to the co-crystal
structures of the complexes. Also, x-ray crystal structures provide a
snapshot of the protein-DNA complex that is constrained by the crystal
lattice, whereas affinity cleavage, while lacking the accuracy of a
crystal structure, serves to illuminate the nature of the nucleotide to
amino acids contacts both in real time and in solution (8). We
generated affinity cleavage maps as described below. Distance
constraints were obtained from the cleavage data and were used to
generate a model of the RTP-bipolar Ter complex as described
in detail in a later section.
Affinity Cleavage Maps--
The residues 56, 59, and 63, belonging
to the
It should be noted that the cleavages are expected to shift either to
the 5' or the 3' side from the location of the iron depending on
which groove of the DNA double helix contacted the iron atom (Ref. 16;
see Fig. 2). Thus residue 74, upon derivatization, produced a cleavage
pattern that was shifted to the 3' side, consistent with the contact of
the
To make sure that the cleavage pattern was specific, we mutagenized the
G residue of the bipolar Ter contacted by amino acid 56 to a
T. This mutation on the bipolar Ter DNA caused the specific cleavage pattern to disappear and generated a more randomized and
dispersed cleavage pattern in the mutant Ter (Fig.
3C), thus supporting the notion that the residue 56 to G
contact was specific. It should be noted that the G contact described
above is known to be necessary for stable binding of RTP to DNA (14).
Residue 59, after derivatization, promoted several foci of cleavage on both strands of DNA (not shown) as contrasted with the simple cleavage
patterns of derivatized 16 and 63 (Fig. 3, A and
B).
Description of the Molecular Model--
As in the previously
published structure of the RTP-unipolar Ter (10), the
residues 16 and 63 of each RTP monomer interacted with the center of
symmetry within each Bipolar Ter subsite (Fig. 4). A, B and
C, D refer to the monomeric subunits of the two dimers of RTP (Fig. 4).
The computational modeling of RTP-bipolar Ter complex is
shown in Fig. 5. The
Whereas subsequent investigations are warranted, it is tempting to
postulate that the bipolar terminus exhibits the bipolar blockage of
the replication fork, because it is composed of two strong-binding
subsites. Examination of the sequence of this terminus reveals that
these two subsites, called core A and core B, show a strong similarity
to the core subsite within the IRI (unipolar Ter)
sequence. The critical guanines found in the core B (IRIB) subsite are
also found here in equivalent positions, suggesting that each subsite
within the bipolar terminus is indeed a "core"-type of subsite.
This strong/strong binding arrangement yields a plausible explanation
for the behavior of this terminus. Also, the pattern of contacts within
both the subsites of the bipolar terminus is symmetrical.
Interestingly, there are no contacts between residue 74 and the DNA in
the border region between the subsites in the bipolar terminus. This is
in contrast to the IRI terminus where residue 74 of the
D-monomer makes several contacts. Whereas the exact reasons
for this difference in binding remains elusive, it does show that RTP
binds to unipolar and bipolar termini differently.
Cooperative Binding of RTP to the Bipolar Terminus--
A series
of previous work has shown that two RTP dimers bind to the normal
unipolar terminus in a cooperative manner (14, 18, 19). First, the
higher affinity core site is filled by one dimer and then the
overlapping, weaker auxiliary site is filled by the second dimer. We
wished to compare the pattern of RTP binding to the bipolar terminus
site with that of the unipolar site. The IRI fragment (Fig.
6, panel A, lane 1)
showed the typical two-step mobility shift in the presence of RTP with
the first shift corresponding to filling of the core site and with the
second shift corresponding to filling of both the core and auxiliary
site (Fig. 6, panel A, lanes 2-12). The fragment
containing the bipolar terminus (Fig. 6, panel B, lane
1) when mixed with RTP also showed a two-step filling of its sites
in a stepwise manner (Fig. 6, lanes 2-12). A very faint
third shift was observed (7), which might be because of aggregation of
more RTP dimers on the site.
Characteristics of in Vitro Replication Fork Arrest by IRI and
Bipolar Termini--
We wished to characterize the bipolar terminus
with regard to its ability to arrest replication forks in
vitro and also to map the points of arrest of the newly
synthesized DNA. Previously we have used a surrogate in
vitro replication system of E. coli (4, 14) to study
the replication fork arrest by RTP because to date, active preparations
of the DnaB homolog of B. subtilis have not been purified by
any laboratory. Moreover, an efficient replication system that operates
in crude extracts of B. subtilis has not been developed to
date. We used four templates in this study. Fragments containing the
IRI (TerI) site in either orientation were cloned in pUC18 plasmid and
the bipolar terminus was cloned in pUC18 and pUC19 in either
orientation with respect to origin of replication to generate four
templates namely pUC18-IRI, pUC18-IRI Rev., pUC18-Bipolar, and
pUC19-Bipolar for study (see "Experimental Procedures").
The plasmid DNA templates were replicated in vitro using the
E. coli cell extracts in the presence or absence of
different concentrations of RTP and the products were analyzed in a
denaturing urea-polyacrylamide sequencing gel. The pUC18-IRI Rev.
template, containing the IRI site in nonfunctional orientation with
respect to the origin, was replicated in the presence of various
amounts of RTP (Fig. 7, panel
A, lanes 1-4). As expected, RTP did not block the
replication fork in this template (Fig. 7, panel A, lanes 2-4). On the other hand replication of the pUC18-IRI
template containing the IRI site in functional orientation with respect to the replication origin, in the presence of various amounts of RTP
showed a distinct termination product (Fig. 7, panel B, compare lane 1 with lanes 2-4). A major band of
about 600 bases and a minor band between 500 and 600 bp were observed.
We have titrated RTP concentrations and have noticed that at high
concentrations of RTP, the intensities of the bands corresponding to
the replication fork arrest in pUC18-IRI template were reduced, either
because of multiple stops or because of overall inhibition of
replication (data not shown).
The pUC18-Bipolar template replicated in the presence of 0.3, 0.6, and
2.4-fold RTP over template concentration showed a major blocked product
smaller than 500 bases representing the leading strand block product
and smaller blocked products of about 400 bases representing the
lagging strand block products (compare Fig. 7, panel C, lane
1 with lanes 2-4, arrows). With high concentrations of
RTP, the intensity of arrested products decreased and a greater number of bands appeared and total replication also decreased (lane 4). The pUC19-Bipolar template was replicated in the
absence (Fig. 7, panel D, lane 1) or presence of
0.3, 0.6, and 2.4-fold RTP (Fig. 7, panel D, lanes
2-4). As in the case of the pUC18-Bipolar template, the
pUC19-Bipolar template also showed replication arrest products. The
major blocked product smaller than 500 bases represents the leading
strand block product whereas minor blocked products of about 400 bases
represent lagging strand block products. At high RTP concentrations,
the number of bands increased and total replication seemed to decrease.
It may be noted that the RTP concentrations needed to show blockage in
pUC18-Bipolar and pUC19-Bipolar templates was lower in comparison to
that needed in pUC18-IRI and pUC18-IR Rev. templates.
We analyzed the labeled in vitro replication products
further to determine the exact nucleotide sequence where replication was arrested in pUC18-IRI, pUC18-Bipolar, and pUC19-Bipolar templates. The pUC18-IRI template containing a unique XbaI site 140 bp
upstream of the IRI site was digested with XbaI. The
pUC18-Bipolar and pUC19-Bipolar templates containing two
PvuII sites as in the case pUC18/19 (at nucleotides 306 and
628) were digested with PvuII enzyme. The digestion products
were run in a denaturing urea-polyacrylamide sequencing gel along with
pUC18-Bipolar sequencing ladder (data not shown). Fig. 11A
shows the nucleotide sequence of IRI, where the leading strand
is arrested by the RTP·IRI complex. The stop site matches the border
of the minimal effective sequence needed to arrest the helicase (14,
15). Fig. 11, panel B shows the nucleotide sequences on both
sides of the bipolar terminus where replication fork is blocked by the
RTP-Bipolar Ter complex.
Contrahelicase Activity at the Bipolar Terminus--
We have shown
previously that RTP arrests replication by inhibiting the activity of
E. coli replicative helicase, DnaB (4, 14). We wished to
investigate the arrest of helicase in both orientations of the bipolar
terminus. M13mp18 and M13mp19 clones containing the IRI site have been
described (4). The M13mp18-Bipolar and M13mp13-Bipolar templates were
hybridized to 5' end-labeled oligonucleotides to generate the forked
helicase substrates and were assayed for DNA unwinding activity of DnaB
in the absence and presence of RTP. The gels were quantitated with a
phosphorimager, and data were plotted as a graph in Fig.
8. As expected, DnaB was arrested by RTP
on the M13mp19-IRI template but not on the template M13mp18-IRI (4,
14). Unwinding was blocked on the M13mp19-Bipolar template (Fig. 8) as
in M13mp19-IRI. However, in the case of the M13mp18-Bipolar template,
RTP blocked oligonucleotide release by about 50% in contrast with only
an ~10% release in the case of M13mp18-IRI. Thus, although the
bipolar terminus blocked replication and helicase activity from both
orientations, one orientation of the terminus blocked the helicase more
efficiently than the other.
We further investigated helicase blockage by the bipolar terminus with
heteroduplex substrates using a technique developed by us (14, 15). The
M13 universal forward primer labeled at the 5' position was annealed to
the four single-stranded DNA templates and was extended by DNA
polymerase (sequenase) in the presence of all four dNTPs and one ddNTP
(Fig. 9A). Four sets of
reactions were carried out, each containing a different ddNTP. Because
equimolar amounts of DnaC stimulate DnaB helicase activity (20), we
performed the helicase assays either with only DnaB or with DnaB in the presence of DnaC. We measured oligonucleotide release by DnaB (or DnaB
plus DnaC) from the heteroduplex substrates in the absence or presence
of different concentrations of RTP. The template M13mp18-Bipolar (Fig.
9B, bottom) showed reduction in DNA unwinding but
not as strongly as in the M13mp19-Bipolar template (Fig. 9B,
top) consistent with the observation that one orientation of
the bipolar terminus was more efficient than the other in arresting the
helicase.
We wished to determine the minimum effective sequence needed to arrest
the helicase at the bipolar terminus in both orientations. Dideoxy
chain termination reactions were carried out separately with both the
M13mp18-Bipolar and the M13mp19-Bipolar templates in the presence of
each of the four dideoxynucleotide triphosphates (ddNTP) and
[
The last group of extension products released (beyond which there was a
sharp disappearance of extension products because of RTP-mediated
blockage of DnaB), was eluted from the gel (Fig. 9B, see
arrow and bracket) and resolved in a denaturing
sequencing gel along with the pUC18-Bipolar and the pUC19-Bipolar
sequencing ladders (Fig. 10,
top and bottom). The precise location of the critical bases in both orientations of the bipolar terminus was determined from the sequencing gels and is shown in Fig.
11.
Transcription Arrest at the Bipolar Terminus--
Previously we
have shown that the RTP·IRI complex can block transcription by T7,
SP6, E. coli, and yeast RNA polymerases in a polar fashion
(11, 13). Because the bipolar terminus blocks replication and DnaB
helicase from both orientations albeit with unequal efficiency, we
wanted to investigate whether, and to what extent, the bipolarity also
extended the arrest of transcription. The fragments containing the
bipolar terminus in either orientation were cloned in pET22b plasmid as
described under "Experimental Procedures." The four pET22b-based
clones containing IRI or bipolar terminus were linearized with BlpI and
transcribed in the absence or presence of RTP.
RTP blocked T7 RNA polymerase-catalyzed transcription at IRI in a polar
fashion (11). The pET22b-IRI (pET22b-BS3) template transcribed in the
absence of RTP, yielded a single major transcript, whereas in the
presence of 0.35, 0.7, or 1.4-fold RTP, a truncated product was formed
with increasing intensity (Fig.
12A, panel A, lanes
1-4). However, in the template pET22b-IRI Rev. (pET22b-BS3 Rev.)
no major truncated transcript was formed in the absence or presence of
RTP (Fig. 12B, lanes 1-4). When pET22b-Bipolar
template was transcribed in the presence of 0.35, 0.7, or 1.4-fold RTP, a truncated transcript was generated (Fig. 12D, lanes
1-4) as in the case of pET22b-IRI. Similarly pET22b-Bipolar Rev.
template showed generation of a truncated transcript in the presence of RTP (Fig. 12C, lanes 1-4) although with less
efficiency than the pET22b-Bipolar template (compare the amount of
full-length transcripts in lanes 2-4 of Fig. 12C
with 12D). The transcription blocking efficiency of IRI and
bipolar templates were quantitated with a phosphorimager and are shown
in Fig. 12E, and the data support the conclusions stated
above.
The first part of the work reported in this study was initiated
with the primary objective of trying to understand the possible molecular basis of the polarity of arrest of the replication forks and
the replicative helicase, by comparing the nucleotide to amino acid
contacts of RTP bound to the bipolar terminus with that of the more
ubiquitous unipolar terminus. We have also constructed a
three-dimensional model of the RTP-bipolar Ter complex by
combining the crystal structure of the apoprotein (5) and the affinity cleavage data. The model that resulted is consistent with mutagenesis and cross-linking data that suggested roles for the Inspection of the bipolar Ter sequence of the plasmid pLS20
of B. subtilis did not provide definitive information on the
molecular basis of polarity. The methylation protection data reported
here did provide a preliminary picture of symmetrical contacts of RTP with the bipolar terminus but did not reveal precise amino acid to base
contacts, which was obtained from the affinity cleavage data. The
affinity cleavage method, used to construct models of DNA-protein
complexes when the crystal structure of the apoprotein is already
known, has proven to be a very useful adjunct to the DNA-protein
co-crystal structures derived by x-ray crystallography (8, 9, 22). This
study has revealed symmetrical base to amino acid contact in the
bipolar Ter, as contrasted with the asymmetric and more
frequent contacts with the core site and fewer contacts with the
auxiliary site that were observed in the more ubiquitous unipolar
Ter (10). This structural analysis strongly suggests that
symmetrical RTP dimers appeared to generate polarity by asymmetric
protein-DNA contacts in unipolar Ter and, in the bipolar
Ter, the symmetric contacts abolished or reduced polarity. In contrast with RTP of B. subtilis, the Tus protein of
E. coli caused polar arrest of helicases because of the
inherent asymmetry of the monmeric protein bound to a single
Ter site (23).
A careful comparison of the RTP-bipolar Ter complex with
that of the RTP-unipolar Ter complex also revealed
that the contacts seen in the two cases are different in some
additional regards. For example, residue 74 that is located in the Does DNA-protein contact directly and by itself promote polarity by
strong DNA binding of RTP to the core and weaker binding to the
auxiliary site, or does the mechanism also involve productive helicase-RTP protein-protein contact on one side and poorer or no
contact at the other site? We believe that DNA-protein interaction is
one factor, but not the only factor, in promoting polarity for the
following reasons. First, there is a large amount of in vitro data that support RTP-DnaB interaction in vitro
(24). Second, we have recently observed that a structurally asymmetric Ter site located near a replication checkpoint of B. subtilis arrests forks in a bipolar fashion, albeit inefficiently
(25). The available data support the idea that appropriate protein-DNA interaction creates a conformation that allows helicase to contact the
contrahelicase domain of RTP. Having a symmetrical Ter
sequence would be one obvious way of achieving that end. It is however possible that different but quasi-equivalent RTP-DNA contacts at the
core and auxiliary sites of a Ter site, such as was observed at or near the left checkpoint of B. subtilis chromosome,
would generate a conformation necessary for RTP-helicase contact on both sides of the Ter site, thus promoting bipolarity of
fork arrest.
The observation (Fig. 6) that the binding of the two dimers of RTP to
the bipolar Ter seems to show cooperativity is consistent with our earlier reports that it is not enough to have two dimers of
RTP merely binding to two tandem core sites to arrest a helicase but
that the two dimers must be interactive (14). It should be noted that
the helicase arrest experiment shows the minimal effective sequence
needed to arrest the helicase from both directions. It does not show
the precise location where the helicase actually stops. The site at
which most of the newly synthesized DNA stop appears to be just inside
the minimum effective sequence needed for helicase arrest. In this
context, it might be useful to keep in mind that the behavior of the
solo helicase at the terminus might not exactly duplicate that of the
helicase associated with the replication complex in
vitro.
Perhaps it is interesting that replication termini also arrest RNA
polymerases in a polar fashion. However, the observation does not
necessarily support the notion that any enzyme that slides on DNA or
melts DNA would be arrested by RTP. Our observations reported
previously (15) showed that several helicases that support rolling
circle-type replication are not arrested by RTP.
The results of the helicase ladder experiment described here showed
that RTP arrested DnaB helicase-catalyzed unwinding of DNA regardless
of the size of the double-stranded region, from a few bases to more
than one kilobase. If RTP merely blocked helicase translocation rather
than authentic unwinding, then it would have been expected to inhibit
only the release of smaller fragments of the ladder rather than the
larger ones (14, 26). Inhibition of unwinding of partially
double-stranded DNA regardless of the lengths of the fragments tends to
be consistent with the idea that RTP arrested authentic DNA unwinding
rather than just the translocation of the helicase.
Finally, the ability of RTP to arrest RNA polymerase and several
helicases might suggest that RTP arrests these enzymes at the
Ter site nonspecifically by binding tightly to DNA. However, we have presented evidence elsewhere that is more consistent with the
notion that RTP-mediated arrest not only involves RTP-Ter DNA interaction but also protein-protein interaction between the arresting and the arrested proteins (24, 25). Recent work from our
laboratory with the Tus protein of E. coli has also
demonstrated a critical role of the terminator protein-DnaB interaction
in fork arrest.3 It appears
likely that although Tus and RTP are structurally different, they
arrest the replicative helicase by the same mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix.
No attempts were made to mutate this residue to a noncysteine moiety.
Instead, additional Cys residues were introduced one at a time into the locations indicated (see Figs. 3 and 4) by site-directed mutagenesis (QuickChange kit, Stratagene), and the residues were derivatized with
EPD and cleavage reactions were performed as published (8, 10).
When DNA cleavage is catalyzed by an Fe-EDTA-conjugated DNA-binding
protein, cleavage occurs at the C1' and/or C4' bonds of the sugar
moieties within 3-4 Å from the location of the hydroxyradical generator, i.e. the iron atom. This iron atom, in turn, is
located at a distance of 14 Å from the
-carbon atom of the
conjugated cysteine (this assumes a fully extended conformation of the
EPD chain). Therefore, the cleavage of the phosphodiester backbone occurs at a net distance of 14 ± 3-4 Å from the
-carbon of
the cysteine. This allowed a distance constraint to be assigned between the locations of the cysteine conjugates on RTP and the corresponding cleavage sites on the DNA. The cleavage data were converted to distance
constraints for use with the X-PLOR program package. These distance
constraints were converted to an energy term for the molecular force
field by being modeled as NOE (Nuclear Overhauser Effect) restraints, a
standard procedure in NMR structure refinement. This procedure does not
implicate a direct energetic relationship between the Fe-EDTA and the
contacted bases. Rather, utilizing the NOE term is a convenient way to
translate the affinity cleavage data into distance constraints that can
be used for automatic construction of the model. The model consists of
two dimers of RTP positioned 15 Å from the axis of the terminus DNA
(modeled as a linear duplex of B form DNA) with the dyad axis
positioned parallel to the center of each of the two subsites within
the duplex. Energy minimization was then used to construct the
protein-DNA model. Although there are several simulation techniques
that could be used to generate this model, the most successful
simulation involved defining each secondary structure feature of the
protein and each planar sugar moiety and base of the DNA as a rigid
body that was allowed six degrees of freedom: three translational and three rotational. The relative positions of the atoms within such a
rigid body are not allowed to change independently. Powell minimization was then used to gradually and reproducibly minimize the overall structure of the simulated structure. To maintain the integrity of the
individual structural elements within the model, the energy terms used
within the force field included van der Waals interactions, electrostatic interactions, hydrogen-bonding potentials, and bond energies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-elimination in sequencing gels. The summary of the comparative methylation protection data is
shown in Fig. 1B. A representative protection data of the
top strand of the bipolar terminus is shown in Fig. 1C.
View larger version (49K):
[in a new window]
Fig. 1.
Comparison of sequences of unipolar (IRI) and
bipolar termini and purine contacts made by RTP. A,
unipolar terminus with a typical core and an overlapping auxiliary site
and the bipolar terminus, with two overlapping core sites. The
filled circles show the homology between the two termini.
B, summary of methylation protection experiments. The
methylation protection data for the unipolar terminus is from Ref. 14.
The RTP dimers contacted G residues that are available for interaction
in the major groove of the DNA. Note that the core site of the unipolar
terminus shows more G contacts than the auxiliary site. Note the
symmetrical distribution of G contacts on the two overlapping core
sites of the bipolar terminus. C, autoradiogram showing
methylation protection data of G residues 4, 13, 20, and 29 of the top
strand of bipolar terminus.
View larger version (23K):
[in a new window]
Fig. 2.
Conversion of RTP to a site-directed chemical
nuclease. A, structure of the
(S)-(2-pyridylthio)-cysteaminyl-EDTA-Fe complex and its
conjugation by exchange reaction with the solvent-accessible cysteine
residues introduced into RTP to yield the RTP·Fe·EDTA complex. Note
that the distance of the iron moiety (Fe) from the
-carbon atom of the derivatized cysteine is 14 Å. B,
cleavage pattern of DNA when Fe is positioned over the major
versus the minor grooves. Note that the cleavage pattern for
major groove contact is displaced to the 5'-end whereas that for minor
groove contact is displaced to the 3'-end, because DNA is a right
handed double helix. Moreover, the hydroxyradicals cleave C1' and C4'
bonds of the sugar moiety that are exposed only in the minor
groove.
3 DNA-recognition helix and the residues 16 and 74 from the
1 helix and the
2 strand, respectively were separately
mutagenized to cysteines, and the resulting mutant forms were coupled
to EPD. The protein-Fe-EDTA conjugates were used to cleave bipolar
Ter DNA that was singly end-labeled at the 5'-ends after
adding ascorbate to the reaction mixture. The cleavage pattern of some
representative derivatives is shown in Fig.
3. Controls are described in the legend
to Fig. 3. Nonderivatized RTP did not cleave DNA either in the presence or in the absence of ascorbate. Wild-type RTP treated with EPD did not
cleave DNA with ascorbate. Derivatized RTP needed ascorbate to generate
hydroxy radicals that in turn cleaved DNA.
View larger version (54K):
[in a new window]
Fig. 3.
Autoradiograms of the Fe-EDTA cleavage of
labeled bipolar terminus DNA fragment. Cysteine residues were
introduced into rationally selected residues (16, 56, 59 (not shown),
63, and 74 (not shown)) of RTP by site-directed mutagenesis and coupled
to Fe-EDTA. Hydroxyradicals were generated by adding ascorbate, as
described. Panel A, cleavage of DNA labeled in the top or
bottom strand with RTP E63C. Panel B, cleavage
of DNA labeled in the top or the bottom strand with RTP R16C.
Panel C, cleavage of DNA labeled in the top or the bottom
strand with RTP E56C (only the cleavage pattern of the top strand is
shown). M and M' are markers generated by G>A
cleavage of the same end-labeled DNA. A and A'
show the cleavage patterns on the bipolar Ter site and of a
mutant site where the G contact (arrow) has been mutated to
a T; note that the specific cleavage seen in A is randomized
in A'. The control lanes M and A-F in
panels A--C are as follows: M, G>A cleavage
ladder; A, labeled DNA + ascorbate; B, DNA + RTP-EPD (derivatized at the indicated amino acid residues) + ascorbate;
C, wild-type RTP-EPD + DNA + ascorbate; D, DNA + rationally derivatized RTP-EPD without ascorbate; E, DNA
plus underivatized RTP + ascorbate; F, same as in
B but with 50-fold molar excess of competing unlabeled
DNA.
2 strands at the minor groove of Ter DNA (Fig.
4). The residues 16 and 63 both contacted
Ter DNA at the center (Figs. 3 and 4). This result is
consistent with the fact that Arg-16 and Glu-63 are located only 9 Å apart in the crystal structure of RTP (5).
View larger version (25K):
[in a new window]
Fig. 4.
Points of contact between amino acid residues
of RTP and nucleotide residues of the unipolar (IRI) and bipolar
termini. The unipolar terminus data are from Pai et al.
(21). A and B refer to the two monomers of one
RTP dimer whereas C and D refer to the monomers
of the second dimer. Panel A, contact points (cleavage
patterns) on the unipolar terminus. Panel B, contact points
on the bipolar terminus.
3 helices of both
dimers are inserted into the major groove of the DNA duplex; the
anti-parallel
-ribbons straddle the phosphate backbone and insert
into the minor groove. The N-terminal arms are also properly positioned for interactions with the phosphate backbone if they were dropped (the
arms are raised by crystal packing forces in the crystal structure and
therefore remain in that position in the model). The major groove is
forced open by the insertion of the
3 helices, and this necessitated
a global compensation in the DNA structure by a shrinkage of the minor
groove. This distortion disrupts the normal base pairing. This
distortion in the DNA predominated near the center of the RTP dimer and
at the ends of the anti-parallel
-ribbon. Overall, there was only a
slight net curvature of the DNA consistent with published results (17).
The distortions to the DNA were more equally distributed throughout the
entire terminus, rather than there being more severe distortion within the core subsite (Fig. 5). A feasible dimer-dimer interaction can be
investigated by rotating one dimer by 8-10 Å around the DNA axis.
View larger version (34K):
[in a new window]
Fig. 5.
Model of the RTP-Bipolar Ter
complex derived from the crystal structure of RTP and the
RTP-chemical nuclease cleavage data. Note that the model depicts
contact between the 3 helices of RTP with the major groove of the
DNA resulting in some distortions of the major groove. The
2 strands
are contacting the minor grooves. The N-terminal arms are poised over
the DNA so that they could contact sugar phosphate groups. The
dimer-dimer interaction because of association of the
2 strands at
the interphase of the two dimers is not depicted in the model. Note
that the core (IRIB) in this figure has also been referred
to as core B and the auxiliary (IRIA) as core A in the
text.
View larger version (80K):
[in a new window]
Fig. 6.
Autoradiogram of a 5% polyacrylamide gel
showing interaction of RTP with IRI and bipolar termini determined by
gel mobility shift assay. Panel A, lanes 1-12
show interaction of IRI fragment (10 fmols) with 0, 20, 30, 40, 60, 80, 100, 200, 300, 400, 500, and 600 fmols of RTP. Panel B,
lanes 1-12 show interaction of bipolar terminus fragment
with 0, 20, 30, 40, 60, 80, 100, 200, 300, 400, 500, and 600 fmols of
RTP. All the lanes in panel A and B had 10 fmols
of 5'-labeled terminus fragment and 5 µg of calf thymus DNA.
View larger version (95K):
[in a new window]
Fig. 7.
Autoradiogram of a 6% polyacrylamide-8
M urea gel showing unidirectional fork blockage
by IRI and bi-directional fork blockage by bipolar terminus in an
in vitro replication assay using an E. coli in
vitro replication system. Panel A, replication
products of pUC18-IRI Rev. Lanes 1-4, reaction products
obtained in the presence of 0, 2.4, 4.8, and 9.6-fold RTP over DNA
template, respectively. Panel B, replication products of
pUC18-IRI. Lanes 1-4 show reaction products obtained in the
presence of 0, 2.4, 4.8, and 9.6-fold RTP over DNA template
respectively. Panel C, replication products of pUC18-Bipolar
plasmid. Lanes 1-4; 0, 0.3, 0.6, and 2.4-fold RTP over DNA
template, respectively. Panel D, replication products of
pUC19-Bipolar template. Lanes 1-4, reaction products
obtained in the presence of 0, 0.3, 0.6, and 2.4-fold RTP over DNA
template, respectively. Panel E, molecular size markers
(M). The arrows indicate bands generated by
replication arrest by RTP.
View larger version (25K):
[in a new window]
Fig. 8.
Quantitative analysis of the extent of DNA
unwinding by DnaB helicase on M13mp19-IRI (unipolar), M13mp18-IRI Rev
(unipolar-reversed orientation), M13mp19-Bipolar, and M13mp18-Bipolar
Rev. partial duplex helicase substrates. Note that the
M13mp18-Bipolar Rev. substrate was less efficient in arresting helicase
activity than the M13mp19-Bipolar substrate. The unipolar (IRI)
substrate shows the expected polarity of helicase arrest.
View larger version (45K):
[in a new window]
Fig. 9.
Ladder assay of arrest of helicase-catalyzed
DNA unwinding at a bipolar terminus. A, diagram showing
the substrate used in the experiment. The substrates were prepared by
annealing the 5'-labeled M13/pUC forward primer to the M13 based
single-stranded DNA templates containing the bipolar terminus in either
orientation. In four separate reactions, a different ddNTP was included
to randomly terminate the extension products. A nested set of extension
products was thus generated. B, autoradiogram of an 8%
polyacrylamide gel showing bi-directional blockage of DnaB by the
bipolar terminus. Left panel (Top): lane
A, M13mp19-Bipolar substrate plus DnaB and ATP; lanes
B-F, same as in A except that 10, 20, 30, 40, 60, and
160 fmols of RTP was included in the reaction mixture, respectively.
Right panel (Bottom): lane G,
M13mp18-bipolar substrate + DnaB +ATP; lanes H-L contained
substrate + DnaB + ATP and 10, 20, 40, 80, and 160 fmols of RTP,
respectively. The band(s) corresponding to the highest molecular size
DNA band released by the helicase were extracted from the gels from
each of the four ddNTP reactions (arrow and
bracket) and resolved in a DNA sequencing gel (see Fig.
10).
-32P]dATP, and the substrates were purified. The DnaB
unwinding activity was carried out in the presence of high
concentrations of RTP to block unwinding almost completely and was
resolved in 8% nondenaturing polyacrylamide gels and autoradiographed.
View larger version (76K):
[in a new window]
Fig. 10.
Determination of the precise minimum
effective bipolar terminator sequence needed in double-stranded form to
block DnaB helicase. In both top and bottom strands A,
C, G, and T are sequencing ladder
lanes. a, c, g, and t
represent the longest extension products unwound by DnaB from the
ladders generated by ddA, ddC, ddG, and ddT, respectively in the
presence of RTP (from a gel such as shown in Fig. 10). Note that the
sequence ladder was generated by using the same primers and templates
that were used to generate the nested set of extension products shown
in Fig. 9. Thus the minimum double-stranded sequence necessary to
arrest the helicase from either direction can be read off from the
sequencing gels. Top and bottom strands refer to the same substrates of
Fig. 9, respectively.
View larger version (20K):
[in a new window]
Fig. 11.
Sequence of IRI (unipolar) and
bipolar termini showing the stop sites for replication fork and the
boundary of minimum effective terminator sequence needed in the
double-stranded form for contrahelicase activity. Bold
horizontal arrows show the directions of replication fork
progression. Vertical down-arrows show the stop sites of
replication fork coming from the right of unipolar and the bipolar
termini. Vertical up-arrows show the stop sites of
replication fork coming from the left. The two stop sites in IRI are
equivalent to the two right-most stop sites on the bipolar terminus.
The filled triangles represent the minimum effective
terminator sequences in double-stranded form needed for contrahelicase
activity. The numbers refer to the G residues that were protected from
methylation (see Fig. 1).
View larger version (26K):
[in a new window]
Fig. 12.
Arrest of T7 RNA polymerase-catalyzed
transcriptional elongation at IRI (unipolar) and the bipolar
termini. A-D, autoradiograms of a 6% polyacrylamide-8
M urea gel showing arrest of T7 RNA polymerase
transcription by RTP-IRI (unipolar) and RTP-Bipolar terminus complexes
in either orientation. Panel A, transcription of pET22b-IRI
template containing IRI (BS3) site in functional orientation.
Panel B, pET22b-IRI Rev. containing IRI in nonfunctional
orientation. Panel C, pET22b-Bipolar Rev. and panel
D, pET22b-Bipolar. In all panels, lanes 1-4 contained
0, 0.35, 0.7, and 1.4-fold RTP over DNA template. Panel E,
quantitations of the truncated (arrested) transcript formation at both
the unipolar and bipolar termini. Note that the bipolar reverse
orientation is somewhat less efficient in arresting T7 RNA polymerase
than the other orientation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 helix, the
2 strand, and the N-terminal arm of RTP in DNA binding (10, 21).
Thus the
3 helix appears to be the recognition helix that invades
the major groove, and the
2 strand makes minor groove contacts.
Unfortunately, attempts to derivatize the N-terminal arm and to
generate affinity cleavage were unsuccessful.
2
strands of the two dimers contacts the central part of the unipolar
Ter, but these contacts are not visible in the bipolar
Ter.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from NIGMS and a merit award from NIAID of the National Institutes of Health (to D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 919-684-3521;
Fax: 919-684-8735; E-mail: basti002@mc.duke.edu.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M010940200
2 S. White, personal communications.
3 S. Mulugu, A. Potnis, J. Taylor, and D. Bastia, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Ter, polar replication termini; RTP, replication terminator protein; bp, base pairs, EPD, (S)-(2-pyridyl-thio)cysteaminyl-EDTA; IRI, inverted repeat I.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bastia, D., and Mohanty, B. K. (1996) in DNA replication in Eukaryotic Cells (DePamphilis, M., ed) , pp. 177-215, Cold Spring Harbor Laboratory Press, NY |
2. | Bussiere, D. E., and Bastia, D. (1999) Mol. Microbiol. 31, 1611-1618[CrossRef][Medline] [Order article via Infotrieve] |
3. | Carrigan, C. M., Pack, R. A., Smith, M. T., and Wake, R. G. (1991) J. Mol. Biol. 222, 197-207[Medline] [Order article via Infotrieve] |
4. |
Kaul, S.,
Mohanty, B. K.,
Sahoo, T.,
Patel, I.,
Khan, S.,
and Bastia, D.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11143-11147 |
5. | Bussiere, D. E., Bastia, D., and White, S. W. (1995) Cell 80, 651-660[Medline] [Order article via Infotrieve] |
6. | Wake, R. G., and King, G. (1997) Structure 5, 1-5[Medline] [Order article via Infotrieve] |
7. | Meijer, W. J. J., Smith, M. T., Wake, R. G., de Boer, A. L., Venema, G., and Bron, S. (1996) Mol. Microbiol. 19, 1295-1306[Medline] [Order article via Infotrieve] |
8. | Ebright, Y. W., Chen, Y., Pendergrast, P. S., and Ebright, R. H. (1992) Biochemistry 31, 10664-10670[Medline] [Order article via Infotrieve] |
9. | Mazzarelli, J. M., Ermacora, M. R., Fox, R. O., and Grindley, N. D. (1993) Biochemistry 32, 2979-2986[Medline] [Order article via Infotrieve] |
10. |
Pai, K. S.,
Bussiere, D. E.,
Wang, F.,
White, S. W.,
and Bastia, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10647-10652 |
11. | Mohanty, B. K., Sahoo, T., and Bastia, D. (1996) EMBO, J. 15, 2530-2539[Abstract] |
12. | Khatri, G. S., MacAllister, T., Sista, P., and Bastia, D. (1989) Cell 59, 667-674[Medline] [Order article via Infotrieve] |
13. |
Mohanty, B. K.,
Sa hoo, T.,
and Bastia, D.
(1998)
J. Biol. Chem.
273,
3051-3059 |
14. | Sahoo, T., Mohanty, B. K., Patel, I., and Bastia, D. (1995) EMBO, J. 14, 619-628[Abstract] |
15. |
Sahoo, T.,
Mohanty, B. K.,
Lobert, M. L.,
Manna, A. C.,
and Bastia, D.
(1995)
J. Biol. Chem.
270,
29138-29144 |
16. | Oakley, M. G., and Dervan, P. B. (1990) Science 248, 847-850[Medline] [Order article via Infotrieve] |
17. |
Kralicek, A. V.,
Wilson, P. K.,
Ralston, G. B.,
Wake, R. G.,
and King, G. F.
(1997)
Nucleic Acids Res.
25,
590-596 |
18. | Lewis, P. J., Ralston, G. B., Christopherson, R. I., and Wake, R. G. (1990) J. Mol. Biol. 214, 73-84[Medline] [Order article via Infotrieve] |
19. | Langley, D. B., Smith, M. T., Lewis, P. J., and Wake, R. G. (1993) Mol. Microbiol. 10, 771-779[Medline] [Order article via Infotrieve] |
20. |
Wahle, E.,
Lasken, R. S.,
and Kornberg, A.
(1989)
J. Biol. Chem.
264,
2469-2475 |
21. | Pai, K. S., Bussiere, D. E., Wang, F., Hutchison, C. A. I. II, White, S. W., and Bastia, D. (1996) EMBO J. 15, 3164-3173[Abstract] |
22. | Bastia, D. (1996) Structure 4, 661-664[Medline] [Order article via Infotrieve] |
23. | Kamada, K., Horiuchi, T., Oshumi, K., Shimamoto, N., and Morikawa, K. (1996) Nature 383, 598-603[CrossRef][Medline] [Order article via Infotrieve] |
24. | Manna, A. C., Pai, K. S., Bussiere, D. E., Davies, C., White, S. W., and Bastia, D. (1996) Cell 87, 881-891[Medline] [Order article via Infotrieve] |
25. | Gautam, A., and Bastia, D. (2001) J. Biol. Chem. in press |
26. |
Hiasa, H.,
and Marians, K.
(1992)
J. Biol. Chem.
267,
11379-11385 |