(Received for publication, August 16, 1995; and in revised form, October 2, 1995)
From the
Replication forks are arrested at sequence-specific replication termini primarily, perhaps exclusively, by polar arrest of helicase-catalyzed DNA unwinding by the terminator protein. The mechanism of this arrest is of considerable interest. This paper presents experimental evidence in support of four major points pertaining to termination of DNA replication. First, the replication terminator proteins of both Escherichia coli and Bacillus subtilis are helicase-specific contrahelicases, i.e. the proteins specifically impede the activities of helicases that are involved in symmetric DNA replication but not of those involved in conjugative DNA transfer and rolling circle replication. Second, the terminator protein (Ter) of E. coli blocks not only helicase translocation but also authentic DNA unwinding. Third, the replication terminator protein of Gram-positive B. subtilis is a polar contrahelicase of the primosomal helicase PriA of Gram-negative E. coli. Finally, the blockage of PriA-catalyzed DNA unwinding was abrogated by the passage of an RNA transcript through the replication terminator protein-terminus complex. These results are significant because of their relevance to the mechanistic aspects of replication termination.
Specific termination sites of DNA replication exist in the plasmid R6K (1, 2, 3) and the bacteria Escherichia coli(4, 5) and Bacillus subtilis(6, 7) . Sites that arrest replication forks have also been reported in eukaryotes such as Epstein-Barr virus(8) , and in ribosomal DNAs of yeast(9, 10) , plant (11) , and humans(12) . The yeast centromeres are also known to arrest replication fork movement(13) .
In prokaryotes, the replication fork arrest is polar and is mediated by the interaction of replication terminator proteins with the terminus sequence(6, 7, 14, 15, 16) .
The replication terminator protein of E. coli (called Ter
or Tus) and that of B. subtilis (called RTP) ()are
polar contrahelicases, i.e. these proteins inhibit the
activities of DnaB helicase of E. coli in only one orientation
of the terminus sequence with respect to the
origin(17, 18, 19, 20) . Although
several groups have reported that Ter protein of E. coli(17, 21) impedes the activity of DnaB but not
that of helicase II, others believe that the terminator proteins are
general road blocks on DNA that block the passage of many if not all
helicases(22, 23, 24) . The resolution of the
question of helicase specificity of replication terminator proteins has
an obvious bearing on the mechanism of the contrahelicase activity.
Blocking of many helicases that are involved in other functions such as
DNA repair, transcription, conjugative transfer of DNA etc., might
imply that the interactions of Ter and RTP with their respective
binding sites called
and IR sequences (or BS3) are all that might
be necessary to elicit polar impedance of helicase activity. In
contrast, helicase-specific block might imply, in addition to
DNA-terminator protein interaction, also helicase-terminator protein
interaction. The crystal structure of RTP of B. subtilis has
been solved at 2.6-Å resolution(25) . The protein has a
postulated tripartite DNA binding domain that envisages contact with
the minor groove of DNA by the N-terminal arm and the
2-
3
pleated sheets and major groove contact by the
3 region of the
protein. The crystal structure suggests an exposed hydrophobic patch
near the
2-
3 region as a possible surface for interaction
with helicases.
The Ter protein also blocks replication forks of
SV40 and antagonizes the helicase activity of the
T-antigen(26, 27) . We have recently discovered that
Ter and RTP interact with DnaB helicase and SV40 T-antigen. ()Hiasa and Marians (21) have reported that
purified Ter, in vitro, can block DnaB translocation but not
authentic unwinding of DNA duplex. The implication is that blocking of
DNA unwinding requires the participation, along with Ter, of other
proteins. Recently, we have discovered that both Ter and RTP are also
polar anti-transcriptases and block RNA chain elongation by E.
coli, T7, and SP6 RNA polymerases. (
)
The present investigation was mainly driven by the need to answer two questions: (i) are the contrahelicase activities of Ter and RTP helicase-specific? and (ii) do the terminator proteins by themselves, without the assistance of other proteins, block authentic helicase-catalyzed DNA unwinding? In addition to answering the two questions posed above, the results presented in this paper also demonstrate that RTP of Gram-positive B. subtilis, impeded the activity of PriA helicase of Gram-negative E. coli. Furthermore, the PriA-blocking activity of RTP was abrogated by the passage of an RNA transcript through the terminus (BS3)-RTP complex.
Figure 1:
SDS-Polyacrylamide gel
showing the purity of the proteins prepared and used in the various
experiments. Lane A, helicase I; lane B, T7 RNA
polymerase; lane C, Rep helicase; lane D, PriA
helicase; lane E, DnaB; lane F, Ter protein; lane
G, RTP; lane M, molecular mass markers. Lanes
A-G contained 10 µg of each
protein.
Figure 4:
Autoradiogram of an 8% polyacrylamide gel
showing response of helicase I-mediated DNA unwinding to increasing
concentrations of Ter protein. Substrate for helicase assay with
extended heteroduplex region across the site in M13 mp18
and
M13 mp19
was prepared as depicted in the figure (top
panel). Lane M`, double-stranded DNA marker; lane
A, mp18
without helicase I and without Ter; lanes
B-E, mp18
with helicase I and increasing concentrations
of Ter (0, 0.2, 0.4, and 0.8 pmol); lane F, mp19
without
helicase I and without Ter; lanes G-J, mp19
with
helicase I and increasing concentrations of Ter (0, 0.2, 0.4, and 0.8
pmol); lane K, mp18
with DnaB only; lane L,
mp18
with DnaB and 0.4 pmol of Ter; lane M, mp19
with DnaB only; lane N, mp19
with DnaB and 0.4 pmol of
Ter. Note there was no blockage of Helicase I activity in either
orientation by Ter in contrast to polar blockage of DnaB activity
(positive controls).
Figure 5: Polar blockage of PriA helicase by RTP-BS3 complex. M13 mp18BS3PAS and M13 mp18BS3revPAS with the RTP binding site in opposite orientations with regard to PriA movement were constructed as depicted in panel A. A 53-mer oligonucleotide complementary to the BS3 site with a 3` overhang was annealed to the single-stranded DNA, and standard helicase assays were performed after binding RTP to the BS3 site. BS3 and BS3rev are two orientations of the replication terminus of B. subtilis. The arrow ending with a vertical line indicates the orientation of BS3 that blocks a given helicase (panel A). Panel B (top): lane A, mp18BS3PAS substrate without PriA and without RTP; lanes B and C, mp18BS3PAS with DnaB and 0 and 0.4 pmol of RTP; lane D, mp18BS3PAS substrate boiled; lanes E-I, mp18BS3PAS with 40 fmol of PriA and increasing concentrations of RTP (0, 0.1, 0.2, 0.4, and 0.8 pmol, respectively). Panel B (bottom): lane A, mp18BS3revPAS substrate without PriA and without RTP; lanes B and C, mp18BS3revPAS with DnaB and 0 and 0.4 pmol of RTP; lane D, mp18BS3revPAS boiled substrate; lanes E-I, mp18BS3revPAS with 40 fmol of PriA and increasing concentrations of RTP (0, 0.1, 0.2, 0.4, and 0.8 pmol). Note polar blockage of PriA by RTP in one orientation (mp18BS3revPAS) and DnaB in the other (mp18BS3PAS). Panel C, quantitation of the data presented in the autoradiogram shown in panel B. The radioactivity present in each band was measured with a PhosphorImager. Note the polar block of PriA of E. coli by the RTP of B. subtilis.
Figure 6:
Autoradiogram of a nondenaturing 6%
polyacrylamide gel showing polar contrahelicase activity of Ter protein
independent of the length of the double-stranded region in a
heteroduplex containing interstitially located Ter-binding site
(). Top, the helicase substrates with M13 mp18
(Ter-active) and M13 mp19
(Ter-inactive) templates were
constructed as described in the figure. Bottom: lane M, double-stranded DNA marker; lane A, boiled
M13 mp18
substrate; lane B, M13 mp18
without Ter and
without DnaB proteins; lanes C-G, M13 mp18
substrate with 400 ng of DnaB and increasing amounts of Ter protein (0,
0.4, 0.8, 1.2, and 1.6 pmol, respectively); lane H, boiled M13
mp19
substrate; lanes I-N, M13 mp19
substrate
with 400 ng of DnaB and increasing amounts of Ter protein (0, 0.4, 0.8,
1.2, and 1.6 pmol). Note that Ter-
complex blocks DnaB-catalyzed
unwinding of heteroduplex DNA containing the Ter binding site in a
polar fashion and is independent of the size of the
oligonucleotide.
Figure 8: Coupled helicase transcription assay to show that PriA helicase block by RTP can be abrogated by RNA polymerase invading the RTP-BS3 complex. An M13 mp18BS3rev-T7Pr substrate was constructed with RTP binding site (BS3) and T7-RNA polymerase promoter oriented such that RTP-BS3 complex was in the blocking orientation with regard to PriA helicase movement and in the nonblocking orientation with regard to RNA polymerase movement on the same heteroduplex substrate. Lane A, substrate with 200 fmol of PriA only; lane B, substrate with 200 fmol of PriA and 100 fmol of T7 RNA polymerase; lanes C and D, substrate with 200 fmol of PriA and increasing concentrations of RTP (1.6 and 2.0 pmol); lanes E and F, substrate with 200 fmol of PriA, 1.6 pmol of RTP, 50 and 100 fmol of T7 RNA polymerase, respectively; lanes G and H, substrate with PriA, 2.0 pmol of RTP, and 50 and 100 fmol of T7 RNA polymerase. Note the drastic blockage of PriA activity by RTP (lanes C and D) and the significant release of the block by RNA polymerase invasion into and movement across the RTP-BS3 complex (lanes E-H).
Figure 2:
Autoradiogram showing the effect of
RTP-B53 complex and -ter complex on the activity of Rep and
helicase I, respectively. The concentrations of helicase I and Rep were
kept constant at 100 fmol (
18 ng) and 400 fmol (
58 ng),
respectively. The substrates were M13 mp19BS3/
and M13
mp18BS3/
DNAs that were heteroduplexed at the BS3 or the
sites with a 5` end-labeled oligonucleotide. Top left panel: lane A, mp19BS3 substrate without RTP and without Rep; lanes B-F, mp19BS3 with 400 fmol of Rep and 0, 0.4, 0.8,
1.2, and 1.6 pmol of RTP respectively; lane G, mp18BS3
substrate without RTP and without Rep; lanes H-L,
mp18BS3 with 400 fmol of Rep and 0, 0.4, 0.8, 1.2, and 1.6 pmol of RTP,
respectively; lanes M-N, mp19BS3 with DnaB and 0 and 0.4
pmol of RTP, respectively. Note that there is no detectable impedence
of Rep helicase activity by RTP-BS3 complex in either orientation,
whereas DnaB activity is impeded. Top right panel, the
radioactivity present in each band shown in the top
left, was quantitated with a PhosphorImager and plotted. The
quantitations confirm the conclusion stated above. Bottom left
panel: lane A, mp19
substrate without Ter and
helicase I; lanes B-H, mp19
with 100 fmol of
helicase I and 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 pmol of Ter,
respectively; lane I, mp18
without Ter and without
helicase I; lanes J-P, mp18
with 100 fmol of
Helicase I and 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 pmol of Ter,
respectively. Note there is no detectable blockage of helicase I
activity by Ter in either orientation of
. Bottom right
panel, the bands shown in the bottom left panel were quantitated
with a PhosphorImager and plotted. The data support the conclusion made
above.
In view of the reports in the literature that terminator proteins block many helicases(18, 24) , we wished to confirm our observations by keeping the concentrations of RTP and Ter constant and changing the ratios of helicase I to the substrate over a wide range. The helicase assays again showed that neither the activity of Rep helicase (molar ratios of 5-40; RTP 40-fold molar excess over DNA substrate) or of helicase I (molar ratio of enzyme over substrate 5-60; RTP present in 40-fold molar excess over substrate) was impeded by RTP (Fig. 3). Similar experiments performed with Ter protein, helicase I, and Rep helicase yielded identical results (data not shown). Thus neither RTP nor Ter were able to impede the catalytic activities of helicase I and Rep helicase over a wide range of experimental conditions.
Figure 3: The activities of both helicase I and Rep helicase are not impeded by RTP. The RTP concentration was kept fixed (RTP:substrate = 40:1), but the Rep and helicase I concentrations were varied over a wide range. Top left and right panels, the activity of Rep helicase was not impeded over a wide range of concentrations of the helicase (molar excess of enzyme to substrate up to 40-fold) in both orientations of the terminus of B. subtilis (BS3). Bottom left and right panels, the activity of helicase I was not impeded by RTP (kept constant at a concentration of protein:substrate = 40:1) over a wide range of concentrations of helicase I (helicase:substrate up to 60:1) in either orientation of the terminus.
We endeavored to test whether Ter (or RTP)
might block the activity of Helicase I on a DNA substrate that had a
heteroduplex region of only a certain length. Using an oligonucleotide
primer and the circular single-stranded DNA of M13 mp18/19 we
generated a population of DNA substrates that contained various lengths
of heteroduplex regions from <100 bp to >2000 bp (Fig. 4, top). We performed helicase assays in the absence and presence
of various concentrations of Ter. DnaB helicase was used as a positive
control. The results showed that whereas DnaB was impeded in a polar
fashion by Ter (Fig. 4, bottom, lanes k and
l), the activity of helicase I was not impeded, regardless of the
length of the heteroduplexed region present in the substrate (Fig. 4, lanes B-E, and G-J), and
in both orientations of the
sequence.
Figure 7:
Autoradiogram showing precise right
boundary of the minimum effective terminator sequence required for Ter
protein to bind and mediate contrahelicase activity with respect to
DnaB. Lanes a, c, g, and t represent extended primer across the site effectively
released by DnaB in the presence of Ter protein (shown by bracket and arrow in Fig. 6). Lanes A, C, G, and T are sequence ladder across
site generated using the same primer used for extension and helicase
assay. Note when chain extension (i.e. heteroduplex region) is
beyond the ``T'' at the 3` end (bracketed) of the
sequence shown in the bottom panel, Ter protein is able to
effectively block DnaB activity in a polar fashion (lanes D-G in Fig. 6).
Although there is general agreement that Ter imposes a polar block on DnaB-catalyzed DNA unwinding, whether or not Ter is also a general helicase blocker has been a debatable issue(17, 18, 21, 24) . The resolution of this issue should have significant implications for the understanding of the mechanism by which terminator proteins block replication forks, i.e. a block imposed strictly by terminator protein terminus-DNA interaction versus specific protein-protein interaction between the replicative helicase(s) and the terminator protein.
We have critically examined this issue using the
replicative primosomal helicase PriA of E. coli and the Rep
helicase and Helicase I of E. coli as representatives of
non-replicative helicases. Non-replicative helicases implies those that
do not participate in symmetric or ``Cairns type'' DNA
replication. It is worth remembering that Helicase I is involved in
conjugational DNA transfer, whereas Rep helicase is involved in rolling
circle DNA replication of the X174 family of
phages(33, 34) .
We have found that RTP of B.
subtilis acted as a polar contrahelicase of PriA, thus
complementing our earlier results with DnaB of E.
coli(19, 20) . In contrast, we detected no
blockage of either Rep or Helicase I by Ter or RTP over a wide range of
terminator-to-substrate and helicase-to-substrate ratios. Thus we could
not confirm an earlier report claiming polar blockage of Helicase I by
Ter protein(23) . On the basis of our results reported in this
paper, we believe that Ter and RTP are specific contrahelicases of the
replicative helicases DnaB and PriA, and in the case of Ter, of T
antigen of SV40(23, 26, 27) . Implicit in
these results is the idea that the arrest of replication forks at the
terminus involves specific interaction between replicative helicase and
terminator protein(s). Consistent with this notion is our recent
discovery that a point mutation at a residue believed to be in the
helicase blocking domain of RTP, as suggested from its crystal
structure(25) , does not interfere with DNA binding but
abolishes the ability of the resultant RTP to block DnaB helicase. ()
A second issue addressed in this paper is whether Ter,
unaided by any other protein, can block authentic, helicase-catalyzed
DNA unwinding. Our results conclusively show that over a wide range of
50 to greater than 1500 bp of duplex DNA, DNA unwinding by DnaB is
abolished by Ter, thus complementing our earlier results with
RTP(20) . Thus we were unable to confirm the conclusions of
Hiasa and Marians (21) that Ter by itself can block helicase
translocation but not authentic DNA unwinding.
We have reported that
invasion of a terminus, in vitro, by an RNA transcript,
released arrested PriA helicase. This observation provides further
support of our proposition that transcriptional invasion of a
replication terminus is detrimental to the termination process. The RNA
transcripts are halted before entering the terminus by the RNA chain
anti-elongation activity of RTP and Ter in a polar fashion.
The Ter protein of E. coli and RTP of B. subtilis bear little or no homology in the primary amino acid sequence, yet
both proteins block the same replicative helicases of E. coli,
namely DnaB and PriA(17, 19, 20) . On the
basis of crystallography of RTP and mutational analysis, there are
already preliminary indications of the surface of RTP that is involved
in helicase blocking. Although Ter protein has been
refractory to crystallization, mutational analysis of its coding region
should enable one to localize the helicase-blocking region of the
protein. Future work will be directed at localizing the RNA
polymerase-blocking domain on RTP and Ter as well as the interaction
surface on DnaB that recognizes Ter and RTP. Such studies should
illuminate further the mechanism of replication termination.