(Received for publication, October 3, 1994; and in revised form, December 21, 1994)
From the
The transcription repair coupling factor (TRCF) of Escherichia coli has the so-called helicase motifs, is a DNA-,
RNA Pol-, and UvrA-binding protein, and is required for the coupling of
repair to transcription. We investigated the potential helicase,
transcription termination, and transcription-repair coupling activities
of TRCF on various substrates. We found that TRCF does not have a
helicase activity on any of the substrates tested. However, the TRCF
releases both RNA Pol and the truncated transcript from a
transcriptional road block caused by a lesion, a ``missing
base,'' or a DNA-bound protein. It does not have any effect on
rho-dependent or rho-independent transcriptional termination. However,
some premature terminations were induced by TRCF at other sites. The
coupling of transcription to repair occurs with supercoiled and relaxed
circular DNA and with linear DNA. However, the coupling with linear DNA
is strongly affected by the length of the DNA and does not occur with
fragments in which the lesion is closer than 90 nucleotides to the 5`
terminus of the template strand. Under transcription conditions the
repair of lesions in the promoter region and up to the eleventh
transcribed base is inhibited even in the presence of TRCF. Stimulation
of repair in the transcribed strand starts at lesions at +15
nucleotides. Stimulation of repair occurs via facilitating the delivery
of the AB
complex to the lesion site by the
TCRF and can be inhibited by excess UvrA which binds to the TRCF off
DNA. In vitro, strand-specific repair is not dependent on the
MutL and MutS proteins which have recently been implicated in
preferential repair in vivo.
Escherichia coli RNA polymerase is blocked by bulky
lesions including pyrimidine dimers in the template strand, and the
stalled complex makes the lesion inaccessible to the nucleotide
excision repair enzyme, the (A)BC excinuclease (Selby and Sancar,
1990). A transcription-repair coupling factor (TRCF) ()encoded by the mfd gene (Selby et al.,
1991) releases the stalled RNA polymerase and promotes the preferential
repair of the transcribed strand (Mellon and Hanawalt, 1989; Selby and
Sancar, 1991, 1993). The sequence of the Mfd protein revealed so-called
helicase motifs found in a number of helicases including the rho
protein from E. coli (Gorbalenya and Koonin, 1993) which has
properties similar to TRCF in terms of dissociating ternary
transcription complexes (see Richardson (1993)). Therefore, we
investigated the effects of TRCF for helicase activity as well as for
activities relating to rho factor such as releasing RNA Pol from pause
sites, from protein blocks, or from ternary complexes formed as a
result of a ``missing base.'' We found that TRCF differs from
rho in many aspects of its action mechanism but acts similarly to rho
on elongation complexes stalled by a ``protein road block.''
The mechanism of transcription-coupled repair was also investigated
by determining the effect of substrate size, the location of the lesion
relative to the promoter, the concentrations of the UvrA, UvrB, and
UvrC subunits, and the presence or absence of MutL and MutS proteins on
the coupling reaction. Our results reveal that coupling cannot occur
before the subunit is released from the ternary transcription
complex and that a lesion must be
90 nt away from the 5` end of the
transcribed strand for transcription-repair coupling to occur.
Furthermore, by conducting the reaction with varying amounts of UvrA,
UvrB, and UvrC proteins it was found that the levels of UvrA and UvrB,
but not that of UvrC, were critical for the preferential repair of the
template strand. Under our in vitro assay conditions neither
MutL nor MutS (which have recently been implicated in strand-specific
repair) are required for transcription-repair coupling.
To study the effect
of TRCF on transcription termination caused by a protein bound within
the transcription unit, we used the pUNC211 template which contains the tac promoter (Thomas et al., 1985). This template
contains an EcoRI site 270-275 bp downstream from the
transcription start point. Binding of the EcoRI-E111Q mutant
(King et al., 1989; Wright et al., 1989) at an EcoRI site blocks transcription causing the formation of a
stable elongation complex (Pavco and Steege, 1990). pUNC211 was
linearized with PvuII, which cleaves downstream from the EcoRI site to give a 465-base runoff transcript. Reactions, 24
µl, were initiated by mixing the template with E111Q (37 µg/ml)
in 40 mM Tris, pH 7.75, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 100 µg/ml bovine serum
albumin. After 10 min at 37 °C, 0.06 unit of RNA Pol (Promega) was
added in 1 µl and incubation continued for 10 min at 37 °C.
Then 25 µl of repair buffer was added to bring MgCl to
4 mM, ATP to 1 mM, GTP and UTP to 200 µM each, CTP to 20 µM,
[
-
P]CTP to 5 µCi/reaction, rifampicin
to 22 µg/ml, and where indicated, TRCF to 270 nM.
Incubation continued for 20 min at 37 °C. Then 50 µl of repair
buffer was added to bring KCl to 1 M, and after 10 min at 37
°C, reactions were stopped and RNA was analyzed on 3.6% sequencing
gels.
The helicase
assay results in Fig. 1A compare TRCF with a bona fide
helicase, the helicase II of E. coli, and the UvrAB complex which
can dissociate oligomers <50 nt from a single-stranded M13 DNA (Oh
and Grossman, 1987). The results in Fig. 1A were
generated using a 17-nt oligomer annealed to M13 phage DNA. As
expected, helicase II releases the 17-mer readily and the
AB
complex releases it with about 1% the
efficiency of helicase II. In contrast, TRCF even at micromolar
concentrations failed to act as a helicase on this substrate.
Figure 1:
Transcription-repair coupling factor
lacks helicase activity. Helicase assays with TRCF and control proteins
were performed with the following substrates. A, a DNA:DNA
duplex consisting of a 17-nt labeled oligonucleotide annealed to M13
DNA. Circles and are results of independent
experiments with TRCF, triangles for HelII and squares for UvrA
B
represent values
averaged from results of independent experiments. B, a DNA:DNA
duplex consisting of a labeled 71-nt DNA annealed to M13, a DNA:RNA
duplex consisting of a labeled 48-nt RNA annealed to M13, and a
DNA:(5`-tail)RNA structure identical to the DNA:RNA duplex except the
RNA possesses an additional 222 nt at its 5` end, and these 222 nt are
not annealed to the DNA. The latter structure is a substrate for the
helicase activity of rho protein (Brennan et al., 1987). C, structures that closely resemble those present in the
transcription bubble and are described in the accompanying paper,
including a DNA bubble:RNA, which consists of a 90-bp DNA duplex with a
central 12-nt region of non-homology (the bubble). One of the strands
in the bubble (the template strand) is annealed to 12 nt of the 3` end
of a 20-nt radiolabeled RNA molecule. The DNA:RNA structure is the same
as the DNA bubble:RNA except the DNA coding strand is absent. -,
no treatment,
, 95 °C for 10 min.
Having failed to detect a helicase activity with the conventional substrate we tested DNA:DNA, DNA:RNA, and DNA:(5`-tail)RNA duplexes as substrates since these more closely resemble the structures acted upon in a stalled transcription complex. Fig. 1B reveals that while helicase II is capable of dissociating these complexes the TRCF is not. We conclude that TRCF is not an RNA:DNA helicase and does not have a rho-like helicase activity on RNA:DNA hybrids with an RNA tail >40-nt long (Brennan et al., 1987). Consequently, we wished to use a synthetic DNA construct even closer in structure to that found in a transcription bubble. We constructed a 90-nt long duplex with a mismatch which created a bubble to which a 20-nt long RNA was hybridized to create an RNA(5`-tail):DNA bubble duplex (Daube and von Hippel, 1992). Fig. 1C reveals that the TRCF failed to release RNA from this structure. We conclude that the TRCF is a protein with the so-called helicase motifs but with no overt helicase activity.
Figure 2: Lack of rho-like activity of TRCF. A, the effect of rho, TRCF, and t-TRCF proteins on transcription of the 1250-bp BstEII fragment from pMC1 is shown. Radiolabeled transcripts were made by a single round transcription protocol. Unless indicated otherwise, reactions were for 20 min in glutamate buffer. The template has both rho-independent (RI) and rho-dependent (RD) transcription termination sites. RT, product of transcribing to the end of the template. TRCF and t-TRCF did not produce termination at rho-dependent or -independent sites when tested in repair buffer (not shown). B, anti-termination effect of TRCF on run-off transcripts and effect of rho protein and mutant TRCF proteins. The approximately 400-bp PvuII fragment of pGFI-I was transcribed in repair buffer in the presence of proteins at the following concentrations: 43 nM rho, 270 nM TRCF, 600 nM C-X, 1.2 µM C-V, 1.9 µM C-P, 700 nM GNT, and 600 nM t-TRCF (Trunc). Reactions and designations on the gel are as in A.
Figure 3: Effect of TRCF on RNA Pol stalled at a protein roadblock. The EcoRI (E111Q) mutant binds to but does not cleave the template, causing blockage of RNA Pol after synthesizing an approximately 270-nt radiolabeled transcript (BL) (Pavco and Steege, 1990). Upon release of EcoRI by high salt, this transcript can be elongated to full-length (FL, 465-nt). If TRCF is added before displacing EcoRI by high salt, the blocked transcript is released by TRCF and can no longer be elongated to full-length (lane 4).
Figure 4:
Coupling is unaffected by topology of the
template/substrate. Standard transcription-repair coupling reactions
were performed using a reconstituted system of purified enzymes (Selby
and Sancar, 1993). The template/substrate was pDR3274, either undamaged
or irradiated with 225 J/m of UV light, and in negative
superhelical form, relaxed form, or linearized. Repair is detected by
incorporation of radiolabel into repair patches. The figure shows
repair synthesis in the transcribed (T) and nontranscribed (N) strands of the BglI-NsiI fragment, which
originate from the tac transcriptional unit and have been
separated on a sequencing gel. The negatively supercoiled plasmid is
the form isolated from E. coli, the relaxed form was obtained
by incubating the supercoiled form with E. coli TopoI, and the
linearized form was made by treatment with EcoRI.
Figure 5:
Template/substrate requirements for
coupling. The 5kilobase pair EcoRI-HindIII fragment
from pDR3274 was 3` endlabeled at the EcoRI site 235-bp
upstream from the transcription start site, isolated, and irradiated
with 225 J/m of UV light. The DNA was then further digested
with BglI to give a 337-bp labeled fragment, or NsiI
to give a 636-bp labeled fragment, or was not digested. Standard
transcription-repair coupling reactions were performed using a
completely reconstituted system with purified enzymes and the incision
reaction products were then resolved on a 3.6% sequencing gel. The lane
marked G is the Maxam-Gilbert G-specific sequencing reaction
product of the fragment. Note that in addition to the dark G bands, C
and A residues give gray bands, and T residues did not give a
band. In Panel A, incision reaction products in lane 3 are aligned with the photoproduct-forming dipyrimidines identified
in lane G, most notably residues 1 through 19 in the
transcriptional unit (3`, +1-TTAACACTCGCCTATTGTT-+19, 5`).
The same alignments are made in the promoter region. The -35
(3`-AACTGT-5`) and -10 (3`-ATATTA-5`) sequences which are bracketed, and the direction of transcription are shown. Panel A of the figure gives a closeup view of incision in the
region of transcription initiation, and illustrates that the coupling
begins with repair of the T<>T dimer at residues 15-16.
Also, transcription inhibits repair and coupling fails to occur in the
region of the promoter. Panel B shows that coupling fails to
occur within 87 residues from the downstream end of the
template/substrate (discernible in lanes 3 and 6).
We were also interested to know if the distance between the lesion and the promoter distal end of the fragment had an effect on transcription-repair coupling, since a short (138 bp) template/substrate did not undergo transcription-stimulated repair (Selby and Sancar, 1993), and DNA binding studies revealed some evidence that the template may wrap around the TRCF during the coupling reaction (Selby and Sancar, 1995). The end-labeled pDR3274 substrate/template was digested with two enzymes to generate fragments with termini 102 and 401 nt downstream from the transcriptional start site. In a transcription/repair reaction (Fig. 5B) with the 5` end of the template strand a distance of 102 bp from the start site, only the incision at +15 is stimulated by the TRCF (lane 3), indicating that there must be a distance of about 87 nt between the lesion and the 5` end of the template strand for coupling to occur. This conclusion is supported by an inspection of lanes 6 and 9, where it is seen that excision of lesions that are within about 90 bp from the terminus of the fragment in lane 6 are not stimulated by the TRCF, but that the repair of the same lesions is stimulated in lane 9 in which the 5` terminus of the template strand is much farther than 90 nt for lesions corresponding to those examined in lane 6. To conclude, on a linear fragment a lesion must be about +15 from the transcriptional start site and 90 nt away from the 5` end of the template strand in order to be subject to transcription-stimulated repair.
We have no available method for
directly measuring the rates of various steps under transcription
enhanced repair conditions. We attempted to test this prediction by
supplementing the cell-free extract which performs transcription-repair
coupling with each of the three proteins (Fig. 6). The following
results are observed. Addition of UvrA at 6 nM stimulates both
transcription-coupled repair in the tac transcriptional unit,
and overall repair in the tet gene, which is not appreciably
transcribed under our reaction conditions (lanes 5 and 6). At higher concentrations of UvrA, coupling is inhibited
while overall repair is still stimulated (lanes 7 and 8). These data are consistent with UvrA being one of the
limiting factors in transcription repair coupling and in excision
repair. At physiological concentrations most of UvrA is in the form of
AB
complex (Orren and Sancar, 1989); by
increasing the UvrA concentration the amount of free UvrA is increased
which now can bind to TRCF and interfere with its coupling function (lanes 7 and 8). This is supported by the fact that
the anticoupling effect of UvrA can be overcome by high TRCF
concentration (lanes 9 and 10). UvrB also increases
the rate of coupled as well as transcription-independent repair when
added to the CFE (lanes 2-4). However, the effect of
UvrB differs from that of UvrA in two important aspects. The
stimulatory effect of UvrB is more pronounced than that of UvrA, and
even at very high concentrations the coupling does occur even though
the difference in the rates of repair of the template and coding
strands is less at high UvrB concentrations. The data is consistent
with the turnover of UvrA as the rate-limiting step in excision repair,
a step that involves dissociation of UvrA from the
A
B
-damaged DNA preincision complex via
reformation of the free A
B
damage recognition
complex. As the rate of damage recognition increases, the difference
between the coupled and uncoupled repair decreases. In contrast,
addition of a high concentration of UvrC has no detectable effect on
the rates of either overall or coupled repair (lane 1)
consistent with the notion that the loading of UvrB onto the damage
site by the A
B
complex, and aided by TRCF at
transcribed regions is the rate-limiting step.
Figure 6: Effect of high concentrations of UvrA, UvrB, and UvrC proteins on coupling. Standard 25-min transcription-repair synthesis reactions were performed as in Fig. 4using UV irradiated, supercoiled pDR3274 as template/substrate, except transcription and repair were carried out using 30 µg of cell-free extract from E. coli AB1157 per 25-µl reaction. Purified UvrA, B, C, and TRCF were added to reactions as indicated. Fragments from the transcribed (TAC) and nontranscribed (TET) genes in pDR3274 are indicated and the template (T) and nontranscribed (N) strands of each fragment are resolved on the gel.
Figure 7:
Strand-specific repair in vitro using mutL and mutS
cell-free extracts. Standard
transcription-repair synthesis reactions were performed as in Fig. 4using UV-irradiated, supercoiled pDR3274 as
template/substrate, except reactions were catalyzed using 1.2 mg/ml
cell-free extract from mutL (ES1484) and mutS (ES1481) strains of E. coli. The strand-specific repair
signal by extracts was enhanced by addition of purified TRCF (lanes
4 and 9). The addition of MutS protein to the mutS
extract had no effect on
strand-specific repair (lane 8) since the ratio of repair
synthesis in the template/coding strand was the same (1.6) in lanes
7 and 8.
Our work reveals that the TRCF is a protein with helicase motifs but no helicase activity under a variety of conditions. It is quite possible that many of the ``motif helicases'' actually do not have helicase activity but do interact with DNA and perhaps also cause local melting and kinking which are important for the ultimate functions of the protein.
The TRCF is the only known protein in E. coli other than rho that is able to dissociate a ternary RNA Pol complex. However, the results presented here show that the two proteins function similarly in only one aspect, dissociating ternary complexes stalled at a protein ``road block'' to transcription. In other aspects of their functions the proteins differ sharply. TRCF does not dissociate ternary complexes formed at rho-dependent terminators when present at physiological concentrations. Furthermore, rho is an RNA binding protein with a strong RNA-DNA helicase activity. In contrast the TRCF has no measurable affinity to RNA and no helicase activity on RNA-DNA duplexes in a variety of structures.
Our data also reveals that the TRCF brings about its
coupling function by increasing the rate of delivery of UvrB to the
damage region and that this effect can be overcome by increasing UvrA
and UvrB but not UvrC concentrations, suggesting that the preferential
repair of the transcribed strand is accomplished by preferential
delivery of UvrB to the damage site with the aid of TRCF. The precise
mechanism of TRCF aided delivery, however, is still not known, although
it appears that the predicted reaction intermediates are quite
transient when wild-type TRCF and conventional reaction conditions are
used. Notably, a stable TRCF-stalled elongation complex was observed
only when t-TRCF was used, TRCF bound stably to DNA only when ATP
hydrolysis was inhibited, and binding of TRCF to the
UvrAUvrB
damage recognition complex
facilitates dissociation of UvrA and UvrB. We have found that coupling
occurs only when the lesion is approximately 15 nt downstream from the
transcriptional start site and approximately 90 nt from the 5` terminus
of the template strand. These findings lead us to conclude that the
TRCF can couple repair to transcription only when RNA Pol is in the
elongation mode and that the TRCF anchors itself downstream from the
transcription stop site to carry out its coupling function. Finally
since extracts from mutL
or mutS
cells are capable of strand-specific
repair, we conclude that in vitro, TRCF is sufficient to
stimulate nucleotide excision repair of lesions in the template strand
that block RNA Pol.