(Received for publication, October 3, 1994; and in revised form, December 21, 1994)
From the
The 130-kDa mfd gene product is required for coupling transcription to repair in Escherichia coli. Mfd displaces E. coli RNA polymerase (Pol) stalled at a lesion, binds to the damage recognition protein UvrA, and increases the template strand repair rate during transcription. Here, the interactions of Mfd (transcription-repair coupling factor, TRCF) with DNA, RNA Pol, and UvrA were investigated. TRCF bound nonspecifically to double stranded DNA; binding to DNA produced alternating DNase I-protected and -hypersensitive regions, suggesting possible wrapping of the DNA around the enzyme. Weaker binding to single stranded DNA and no binding to single stranded RNA were observed. DNA binding required ATP, and hydrolysis of ATP promoted dissociation. Removal of a stalled RNA Pol also requires ATP hydrolysis. Apparently, TRCF recognizes a stalled elongation complex by directly interacting with RNA Pol, since binding to a synthetic transcription bubble was no stronger than binding to double stranded DNA, and binding to free RNA Pol holoenzyme and to initiation and elongation complexes in the absence of adenosine 5`-O-(thiotriphosphate) were observed. Structure-function analysis showed that residues 379-571 are involved in binding to a stalled RNAP. The helicase motifs region, residues 571-931, binds to ATP and duplex polynucleotide (DNA:DNA or DNA:RNA). Dissociation of the ternary complex upon hydrolysis of ATP also requires the carboxyl terminus of TRCF. Finally, residues 1-378 bind to UvrA and deliver the damage recognition component of the excision nuclease to the lesion.
In Escherichia coli the template strand of an actively
transcribed gene is repaired at a faster rate than the coding strand
(Mellon and Hanawalt, 1989). This phenomenon requires active
participation of the transcription-repair coupling factor (TRCF), ()a 130-kDa protein (Selby and Sancar, 1991, 1993) encoded
by the mfd gene (Witkin, 1966, 1994; Selby et al.,
1991). In a completely defined system, consisting of
template/substrate, RNA Pol, UvrA, B, C, and D, DNA Pol I, DNA ligase,
NAD, and the 4 rNTPs and 4 dNTPs, it was demonstrated that the
nucleotide excision repair enzyme, (A)BC excinuclease, repaired the
template strand preferentially when the TRCF was included in the
reaction mixture (Selby and Sancar, 1993). A model was proposed whereby
the TRCF bound to a stalled RNA Pol, dissociated the stalled RNA
polymerase and the truncated transcript, and delivered the
A
B
complex to the damage site. In this study,
we have investigated the interactions of the TRCF and mutant TRCF
constructs with the various components involved in transcription-repair
coupling in order to correlate the structure of this protein with its
function.
The 138-bp psoralen monoadduct-containing template/substrate and procedures for DNase I footprinting of the stalled elongation complex were as described previously (Selby and Sancar, 1993).
Dr. M. J. Chamberlin generously provided pAR1707, and pDR3274 is as described in Thomas et al.(1985).
The mfd gene was originally cloned as a Sau3A partial digestion product into the BamHI site of pBR322. It was subcloned as an approximately 6-kbp NheI (from pBR322)-SphI (from the insert) fragment into pIBI25 digested with XbaI and SphI to create pMFDONE. The entire mfd gene was further subcloned on an approximately 5-kbp SphI-SspI fragment into the HincII-SphI sites of pIBI24 and pIBI25 to create pMFD102 and pMFD19, respectively. The BamHI-BglII fragment of pMFDONE encoding the first 938 amino acids of Mfd was ligated into the BamHI site of pIBI24. The construct with mfd in the same orientation as lacZ, called pMFD13, encodes t-TRCF (``trunc'' in the bottom of Fig. 1).
Figure 1: Schematic diagram of TRCF and its deletion and fusion constructs. Indicated on the dark horizontal line that represents the Mfd protein are the locations of the UvrB homology region, helicase motifs, and potential leucine zipper (LZ). Restriction sites shown correspond to sites in the gene that were used to construct mutant proteins. C-P, N-V, C-V, N-X, and C-X represent constructs in which the indicated portion of Mfd is fused to MBP. t-TRCF (trunc) is a TRCF deletion mutant that is not fused to MBP.
Constructions for making Mfd deletions fused to the COOH terminus of
maltose-binding protein (MBP) utilized pMAL-c2 (New England Biolabs)
and were as follows. The Mfd component of these fusion proteins are
shown in the top of Fig. 1. pMALMFD(C-V), which encodes amino
acids 571-1148 of Mfd(C-V), was made by inserting the EcoRV-HindIII fragment from pMFD102 into the XmnI-HindIII site of pMAL-c2. pMALMFD(C-X), which
encodes amino acids 379-1148(C-X), was made by inserting the XhoI-HindIII fragment from pMFD102 into the SalI-HindIII site of pMAL-c2. pMALMFD(C-P), which
encodes amino acids 896-1148(C-P), was made by inserting the PvuII-HindIII fragment from pMFD102 into the XmnI-HindIII site of pMAL-c2. To make
carboxyl-terminal deletions, pMFD102 was first changed to pMFDNCO by
site-directed mutagenesis by creating an NcoI site at the
translation start site. Incidentally, this changes the second amino
acid of Mfd from proline to alanine. pMALMFD, in which the entire mfd gene is fused in-frame with the carboxyl terminus of the
MBP gene, was created by inserting the NcoI-HindIII
fragment from pMFDNCO, in which the NcoI end was made blunt by
the Klenow fragment of DNA polymerase I, into pMAL-c2 digested with EcoRI and HindIII, and in which the EcoRI
end was also made blunt. The unique EcoRI site is retained in
pMALMFD. pMALMFD(N-X), which encodes amino acids 1-378(N-X), was
made by inserting the EcoRI-XhoI fragment from
pMALMFD into the EcoRI-SalI site of pMAL-c2.
pMALMFD(N-V), which encodes amino acids 1-571(N-V), was made by
inserting the BglII-EcoRV fragment of pMALMFD into
the BglII-XmnI site of pMAL-c2. All carboxyl-terminal deletion
mutants including t-TRCF were made with the lacZ -complementation
polypeptide in-frame with the carboxyl terminus of the mfd mutant. A K634N mutation at the Walker A GKT sequence was
generated by site-specific mutagenesis and the mutant protein is
referred to as GNT. The wild-type TRCF and the GNT and t-TRCF mutants
were purified as described previously (Selby and Sancar, 1993). The MBP
fusion proteins were purified by chromatography on columns of amylose
resin (New England Biolabs). Uvr proteins were purified as described by
Thomas et al.(1985). Proteins were over 95% pure.
The pull-down assay binding buffer was 1.1 mM KHPO
, 7.7 mM NaH
PO
, 120 mM NaCl, 2.7 mM KCl, 5 mM MgCl
, 10 mM dithiothreitol, 0.002% Nonidet P-40, pH 7.5. Before binding, the
resin was washed and then incubated with 1 ml of binding assay buffer
plus 50 µg/ml BSA for 30 min at 4 °C with gentle rocking. The
resin was then pelleted (in a microcentrifuge at 2500 rpm for 5 min),
the supernatant was removed, and 200 µl of binding buffer (without
BSA) was added. UvrA (1.1 µg) or RNA Pol (1.3 µg) was then
added and mixed with the resin for 45 min at 4 °C. The resin was
then pelleted and the ``free,'' unbound fraction was removed.
The resin was then washed 3 times with 1 ml of binding buffer and the
bound UvrA or RNA Pol was eluted by washing twice with 100 µl of 1 M KCl, 25 mM Tris, pH 7.5, 2 mM dithiothreitol, 1 mM EDTA, and 0.002% Nonidet P-40. The
two bound fractions were pooled. Free and bound fractions were mixed
with 40 µl of sample loading buffer, heated at about 95 °C for
35 min, and resolved on 9 or 10% SDS-polyacrylamide gels. Gels were
stained with silver and bands were quantitated by scanning
densitometry.
Figure 2: Polynucleotide structures utilized. The relative sizes of the DNA, RNA, and bubble are not to scale. The DNA duplex (double stranded DNA) is 95 bp in length. The DNA bubble is a 90-bp DNA duplex with an internal 12-nucleotide region of nonhomology (the bubble). DNA bubble:RNA is the same DNA duplex but with a 20-nucleotide RNA with 8 nucleotides at the 5` end free and the remaining 12 nucleotides annealed to one strand of the bubble. DNA:RNA is the pair of strands in the DNA bubble:RNA structure in which the DNA and RNA are annealed. ssDNA and ssRNA, single-stranded 90-nucleotide DNA and ss 20-nucleotide RNA, respectively.
Binding of Mfd proteins was done in repair assay
buffer (40 mM Hepes, pH 7.8, 100 mM KCl, 8 mM MgCl, 2 mM ATP, 4% glycerol, 5 mM dithiothreitol, and 100 µg/ml BSA) at room temperature for 25
min. For analysis, gel retardation was performed with 5% polyacrylamide
gels in TAE plus 8 mM magnesium acetate. Alternatively, the
DNA was digested with DNase I and analyzed on 6% sequencing gels.
Figure 3:
Binding of TRCF to DNA requires ATP, and
ATP hydrolysis dissociates the complex. Binding of WT and K634N(GNT)
mutants in the absence or presence of 2 mM ATP and 2 mM ATPS were analyzed by gel retardation on a 5% polyacrylamide
gel. Complete retardation occurs in the presence of ATP
S with both
proteins. With the GNT mutant partial retardation is observed with ATP
alone (lane 8).
Figure 4:
ATP hydrolysis is required for removal of
stalled RNA Pol by TRCF. A 138-bp DNA duplex with a psoralen monoadduct
located in the template strand downstream from a tac promoter
and with the template strand radiolabeled at the 5` end was constructed
as described previously (Selby and Sancar, 1993). This template was
transcribed with E. coli RNA Pol in the presence and absence
of 2 mM ATPS and 87 nM TRCF as indicated, and
then the templates were digested with DNase I and the resulting
fragments were resolved on an 8% sequencing gel. The bracket locates the footprint of the stalled elongation complex that forms
when RNAP is blocked at the psoralen-adducted thymine residue (circled T). All reactions contained 2 mM ATP.
The affinity of TRCF to other nucleic acid structures
which may be intermediates during TRC was then investigated in the
presence of ATPS using the gel retardation assay. The results,
presented schematically in Fig. 5, show that TRCF bound most
strongly to and with nearly comparable affinity to all of the nucleic
acid structures having duplex regions. There was an intermediate
affinity for ssDNA, and TRCF did not bind detectably to ssRNA, although
annealing of the ssRNA to the ssDNA to create a 12-bp hybrid region did
enhance the binding to a level comparable to that of the other
duplexes. Thus, the nucleic acid structures that comprise the
transcription bubble do not appear to account for the specificity of
the interaction of TRCF with a stalled RNA Pol. The failure to bind
ssRNA is of special relevance because the rho protein from E.
coli, which also releases a stalled RNA Pol from DNA, is a strong
RNA binding protein and this property of rho is essential for
performing its transcription termination function (Richardson, 1993).
Figure 5:
Binding of TRCF to nucleic acids of
different primary and secondary structures. All binding reactions were
conducted in the presence of 2 mM ATPS. Each
polynucleotide or polynucleotide complex was present at 0.3
nM. Gel retardation was used to separate TRCF-free from bound
polynucleotide. The free and bound polynucleotide were quantified by an
AMBIS scanner.
Figure 6:
DNase I footprint of t-TRCF on DNAs of
various lengths. A 2.1-kilobase fragment of pDR3274 (Selby and Sancar,
1991) was 3`-labeled at the EcoRI site and used directly for
footprinting (lanes 1 and 2) or digested with the
indicated enzymes to generate shorter 3`-labeled fragments which were
then footprinted. The binding reactions contained 2 mM ATPS and 0.5 µM t-TRCF. The digestion products
were separated on a 6% polyacrylamide sequencing gel. Brackets indicate areas of protection against DNase I and asterisks indicate DNase I hypersensitive sites. The same DNase I
footprinting pattern was observed with wild-type TRCF (not
shown).
Figure 7:
Binding of TRCF and its mutant derivatives
to DNA in the presence or absence of ATPS. The binding in the top
panel (A) was conducted with 2 mM ATP + 2 mM ATP
S; in the bottom panel (B), ATP
S was
omitted. Binding was analyzed by gel retardation on 5% polyacrylamide
gels. Proteins were each present at 500 nM in the binding
reactions. The mutant designation is as in Fig. 1. WT-MBP indicates TRCF full-length protein fused to MBP and GNT indicates TRCF with the K635N mutation. Note that in the absence
of ATP
S only the ATPase active site mutant retards the DNA, and
even in this case retardation is not as pronounced as in the presence
of ATP
S because of the residual ATP hydrolysis by the mutant (lane 5).
Figure 8:
Binding of RNA polymerase to TRCF and its
derivatives as measured by pull-down assay. Panel A shows the
binding of 2.9 pmol of RNA Pol or 11 pmol of UvrA to 50 µl of
amylose resin linked to 98 pmol of either MBP or full-length TRCF-MBP
fusion protein. The free (F) and bound (B) fractions
from each binding reaction are shown. Molecular size markers (M) are shown on the right, in kilodaltons, and the
positions of the RNA Pol subunits, UvrA and BSA, are indicated on the left. The BSA present in the free fractions is residual
material left over after preincubating the resins with BSA to occupy
nonspecific protein binding sites. Due to contamination of the free and
bound fractions with small amounts of resin, the MBP and TRCF-MBP
proteins can also be seen on the gel. TRCF-MBP co-migrates with `.
In panel B, binding assays employed 2.9 pmol of RNA Pol
applied to 50 µl of amylose resin linked to 98 pmol of MBP, C-P,
N-V, C-X constructs or full-length TRCF-MBP (WT-MBP). Note again minor
contamination of both free and bound fractions with each MBP
construct.
We also examined the interaction of the TRCF constructs with DNA-bound RNA Pol. We formed stalled elongation complexes by the ``nucleotide starvation'' method (Levin et al., 1987; Krummel and Chamberlin, 1992). In this procedure RNA Pol initiates transcription normally but forms a stalled ternary complex with a 20-nt transcript. Elongation ceases due to a lack of UTP, the 21st nucleotide in the transcript. The ternary complex and the initiation complex are very stable and can be resolved by gel retardation (Levin et al., 1987; Krummel and Chamberlin, 1992), as indicated in Fig. 9A, lanes 2 and 4. An interesting result was seen when t-TRCF was incubated with the initiation and elongation complexes. The t-TRCF protein binds stably to both complexes, producing the supershifts in lanes 3 and 5. Further investigation with the initiation complex, which can be formed without ATP, indicated that the supershift did not require ATP, as shown in Fig. 9B.
Figure 9: A, binding of t-TRCF to initiation and elongation complexes of RNA Pol. The binary and elongation complexes were obtained by omitting or including ApU and three of the rNTPs in the reaction mixtures and analyzed as described by Levin et al.(1987) and Krummel and Chamberlin(1992). Omission of UTP from the reaction prevented transcription beyond 20 nucleotide. Note the retardation of the binary complex in lane 3 and of both the binary and elongation complexes in lane 5 by 640 nM t-TRCF. B, the binding of t-TRCF to the initiation complex is independent of ATP. Note the retarded bands in both lanes 4 and 6. Only the region of the gel containing the retarded complexes is shown. C, identification of RNA Pol binding domain of TRCF. To RNA Pol/template mixtures the indicated TRCF or its derivatives were added to 500 nM and the products were analyzed by gel retardation. Only the upper half of the gel is shown. WT-MBP, full-length TRCF-MBP fusion protein.
The various TRCF constructs were assayed for their ability to stably bind initiation and elongation complexes in the gel shift assay. The results, given in Fig. 9C, show that stable binding was observed only with t-TRCF (lane 4). The negative results with the N-V construct (lane 3), which did bind to free RNA Pol in the pull-down assay, and with the GNT protein (lane 5), which might be expected to be able to bind to but not remove the stalled RNAP, probably arise from the relatively harsh electrophoretic separation conditions of the gel retardation assay compared to conditions for binding to affinity resins in solution. Interestingly, the other deletion construct that bound RNA Pol in the pull-down assay, C-X (lane 8), was able to dissociate the stalled RNA Pol as completely as wild-type TRCF (lane 9). This result indicates that residues 1-378 are not required for this reaction with stalled RNA Pol. On the other hand, the failure of C-V (lane 7) to remove the stalled RNA Pol indicates that residues 379-571 are essential for this process. Finally, the observation that t-TRCF binds but does not dissociate the ternary complex suggests that residues 896-1148 in the carboxyl terminus may be required for release of RNA Pol from elongation complexes upon hydrolysis of ATP.
Figure 10: Identification of the UvrA binding domain of TRCF. Pull-down assays employed 11 pmol of UvrA applied to amylose resin linked to 18 pmol of MBP or the indicated MBP-TRCF fusion construct. WT-MBP is the full-length TRCF-MBP fusion protein. The UvrA band is indicated. Each MBP construct is present in the bound and free fractions due to contamination of the fractions with a small amount of resin. For example, some C-X protein can be seen in lanes 7 and 8. The N-V construct (lanes 11 and 12) migrates only slightly faster than UvrA, and the presence of UvrA in the N-V bound fraction is evident upon close inspection of the gel.
Interestingly, when the reciprocal binding experiments were conducted, that is, when TRCF or its truncated derivatives were applied to a UvrA affinity column they did not bind to the column. The only exception was the N-X (residues 1-378) construct which bound efficiently to the UvrA column (data not shown). Failure of reciprocal binding in protein affinity chromatography is a common occurrence and it is usually attributed to steric factors (see Formosa et al.(1991)). Thus, it is conceivable that the large deletion in N-X makes this domain more accessible to UvrA immobilized on a resin.
While subcloning mfd we made the observation that the construct which encodes the
t-TRCF made cells hypersensitive to UV by a dose modification factor of
3. Since the host cell line, UNCR9F`laci (mfd
recA
) was uvr
, it seemed possible that t-TRCF inhibited
nucleotide excision repair. We tested this possibility by performing
excision repair in vitro using wild-type cell-free extracts in
the presence and absence of purified t-TRCF. The results in Fig. 11show that t-TRCF inhibits both transcription-independent
repair (lanes 3 and 4) and transcription-dependent
repair (lanes 5 and 6). The inhibition of
transcription-dependent repair follows from our observation that t-TRCF
binds stably to the stalled elongation complex and thus prevents the
native TRCF present in the extract from performing its function. Since
TRCF binds to UvrA, it seemed likely that the inhibition of
transcription-independent repair resulted from an interaction of t-TRCF
with this subunit. This prediction was tested using the gel retardation
assay to examine the binding of UvrA to a radiolabeled, UV-irradiated
DNA fragment. The results in Fig. 12A show that t-TRCF
prevents the binding of UvrA to damaged DNA, both in the absence (lanes 1-8) and presence (lanes 9-12) of
unlabeled, plasmid DNA. In contrast, Fig. 12B shows
that wild-type TRCF had no effect on UvrA binding to damaged DNA.
Furthermore, the inhibition of repair by t-TRCF shown in Fig. 11could be overcome by supplementing the reactions with
purified UvrA, and inhibition of strand-specific repair could be
overcome by adding UvrA and TRCF (data not shown). Thus t-TRCF appears
to bind to UvrA in a manner different from wild-type TRCF, and t-TRCF
effectively traps this Uvr subunit, making it unavailable to catalyze
repair.
Figure 11: Inhibition of nucleotide excision repair by t-TRCF (Trunc). Standard transcription-repair synthesis reactions were performed with cell-free extracts from AB1157 and UV-irradiated pDR3274 as the template/substrate. UV-dependent repair was measured as incorporation of label into the DNA. The repaired DNA, digested with the appropriate restriction enzymes, was resolved on a 3.6% sequencing gel to separate the template (T) and nontranscribed (N) strands of fragments from the strongly transcribed tac transcriptional unit (TAC) and from the inactive tet gene (TET). Rifampicin was used in lanes 3 and 4 to inhibit transcription. t-TRCF inhibited both transcription-dependent and -independent repair.
Figure 12:
Inhibition of UvrA binding to damaged DNA
by t-TRCF (Trunc). Gel retardation was used to demonstrate UV
damage-dependent binding of UvrA to an approximately 300-bp
radiolabeled DNA fragment. Part A shows that t-TRCF (Trunc) inhibits the binding of UvrA to damaged DNA both in
the absence (lanes 1-8) and presence (lanes
9-12) of 200 ng of pBR322. Part B shows that
wild-type TRCF does not effect UvrA binding to DNA. ATPS was not
included in the binding reactions.
The current findings with reference to sequence motifs in and functional properties of TRCF are discussed here. Fig. 13summarizes the correlations that we have identified between functional properties and regions of the Mfd protein.
Figure 13:
Schematic diagram of structural and
functional domains of TRCF. The primary structure of the 1148-residue
Mfd protein (E. coli TRCF) is indicated with the horizontal line interrupted with open boxes which
locate the region of sequence similarity to UvrB (UvrB
homology) and the helicase motifs. Regions of the protein
associated with various functions are indicated above the
protein with brackets. Binding of the helicase motif regions
of TRCF to DNA requires binding of ATP; hydrolysis of ATP causes
dissociation of the helicase motif region from DNA. Hydrolysis of ATP
is also required for dissociation of a stalled RNA Pol; therefore, the
DNA-dissociation activity of the helicase motifs is probably required
for removing a stalled RNA Pol. Residues 939-1148 in the carboxyl
terminus are also required for dissociating a stalled RNA Pol since the
t-TRCF protein which lacks these residues binds to but does not
dissociate the stalled RNA Pol. The lack of the carboxyl terminus in
t-TRCF also produces a higher affinity of this mutant for DNA, and
appears to result in a more stable binding to free UvrA. The UvrB
protein has two UvrA binding sites (see Footnote 3). In the
UvrAUvrB
damage recognition complex, UvrB
may partially cover the site on UvrA that the amino terminus of TRCF
contacts. Thus when TRCF dissociates RNA Pol and binds UvrA
UvrB
to aid in damage recognition, binding of the
amino terminus of TRCF to UvrA may facilitate dissociation of UvrA and
formation of the UvrB-damaged DNA preincision complex.
Whether the finding that TRCF binds to RNA Pol holoenzyme has any
physiological relevance cannot be ascertained at present. However, it
does have a structural significance: it leads to the conclusion that
the subunit does not interfere with the binding of TRCF to RNA
Pol. It also suggests that the binding of TRCF to stalled RNA Pol is
not dependent on the special conformation of RNA Pol in the elongation
complex.