©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Function of Transcription-Repair Coupling Factor
I. STRUCTURAL DOMAINS AND BINDING PROPERTIES (*)

(Received for publication, October 3, 1994; and in revised form, December 21, 1994)

Christopher P. Selby Aziz Sancar

From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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), (^1)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(2)B(1) 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.


MATERIALS AND METHODS

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).

Cell Lines and Plasmids

E. coli AB1157 was used as the wild-type cell line with regard to repair. This strain was obtained from the E. coli Genetic Stock Center, Yale University. A knock-out mutant deleted in mfd was constructed from AB1157 by the method of Washburn and Kushner(1991). In the resultant construct, UNCNOMFD, a 2317-bp NsiI-NsiI fragment of mfd was replaced by the PstI-PstI kanamycin resistance-encoding DNA fragment from pUC4K (Pharmacia Biotech Inc.). UV survival was measured in UNCR9F`laci^Q, a recA mfd derivative of AB1157 (Selby and Sancar, 1993). Mfd mutant proteins were expressed in UNCR9F`laci^Q and fusion proteins were expressed in DH5alphaF`laci^Q.

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 alpha-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.

Protein-Protein Interaction

The binding of TRCF constructs to UvrA and RNA Pol was by ``pull-down'' assay. DH5alphaF`laci^Q cells were transformed with each of the MBP-TRCF fusion constructs. The fusion protein was overproduced by adding isopropyl-1-thio-beta-D-galactopyranoside to 0.3 mM to cultures at an OD of approximately 0.6, and further incubating for about 6 h. Cell-free extract was made from each and several milligrams of total cellular protein were mixed with 0.5-1.0 ml of amylose-agarose resin in 10-50 ml of ``column buffer'' (25 mM Tris, pH 7.5, 200 mM NaCl, 1 mM EDTA, and 2 mM dithiothreitol) by gentle rocking for 3-4 h at 4 °C. The resin was washed three times with 10-50 volumes and then stored in the same buffer. A sample of each resin was eluted with column buffer containing 10 mM maltose and the protein was quantitated by Bradford assay. Samples of each resin were also resolved on SDS-polyacrylamide gels to confirm the quantitation of bound protein and to ascertain its purity. The resin-bound fusion proteins were over 90% pure. The prepared resins were mixed with equilibrated amylose resin in the appropriate proportions in order to control the ratio of fusion protein to resin.

The pull-down assay binding buffer was 1.1 mM K(2)HPO(4), 7.7 mM NaH(2)PO(4), 120 mM NaCl, 2.7 mM KCl, 5 mM MgCl(2), 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.

Binding to Polynucleotides

The polynucleotide structures that we used are illustrated in Fig. 2, and most were based on the synthetic, stable RNA:DNA bubble duplex devised by Daube and von Hippel(1992). Their synthetic transcription bubble is comprised of an 80-bp DNA duplex with an internal 12-base region of non-homology (the bubble). A 20-nucleotide RNA is annealed to this duplex such that 8 bases at the 5` end of the RNA are free and the 12 bases at the 3` end are annealed to one strand of the DNA in the bubble. Our RNA and DNA molecules were identical to those described (Daube and von Hippel, 1992) except the duplex region of the DNA is 10 bp longer upstream of the bubble. Either one of the DNA strands or the RNA was labeled with P at the 5` end, and oligonucleotides were annealed to give the DNA:DNA bubble duplex, the RNA:DNA hybrid, or the complete DNA:DNA:RNA synthetic transcription bubble. A 95-bp DNA duplex was made by annealing two complementary oligonucleotides, one of which was labeled at the 5` end. DNA was synthesized by Operon Technologies, Inc. and RNA was from Oligos etc. An additional DNA duplex was generated by labeling the 3` end of the unique EcoRI site in pDR3274 and gel purifying the 2.1-kbp EcoRI-BglII fragment. This DNA was subsequently digested with NruI, NsiI, BstYI, BglI, or SspI to generate 3` end-labeled fragments 982, 644, 476, 344, or 184 bp in length, respectively.


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), 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.

Interaction with Stalled RNA Pol

The polymerase chain reaction was used as described by Krummel and Chamberlin(1992) to synthesize from pAR1707 a 5`-end labeled DNA fragment which contains the T7 A1 promoter. This approximately 300-bp fragment was gel purified and used to generate ``template 1 A20 complexes'' (Krummel and Chamberlin, 1992), which consist of E. coli RNA Pol stalled 20 bases into the transcriptional unit due to nucleotide (UTP) starvation (Levin et al., 1987; Krummel and Chamberlin, 1992). Stalled complexes were formed in two steps. First, the template was mixed in 25 µl with RNA Pol (6.5 times 10 units, Promega) and Mfd protein construct (500 nM) in repair assay buffer. After 12 min at 37 °C, ApU (the first dinucleotide in the transcript) was brought to 100 µM, CTP to 10 µM, GTP to 10 µM, and rifampicin to 22 µg/ml by adding these components in 25 µl of repair assay buffer. After 30 min at 37 °C, the reaction mixtures were loaded onto 34-cm long 4.5% polyacrylamide gels and run in TBE buffer in a 4 °C cold room at 100-200 volts for about 40 h.

Strand-specific Repair Synthesis Assay

This assay was performed as described (Selby and Sancar, 1993). UV-irradiated superhelical pDR3274 was incubated with wild-type cell-free extract from AB1157 which performs transcription, repair, and transcription-coupled repair. Repair was measured as the incorporation of label into the template and coding strands, after the DNA was digested with the appropriate restriction enzymes and fragments were resolved on a 3.6% sequencing gel.

Gel Retardation Assay of UvrA Binding to Damaged DNA

The substrate was the approximately 300-bp radiolabeled fragment amplified from pAR1707 as described above. Irradiation was with 700 J/m^2 of UV light. Substrate was mixed with 7 nM UvrA in 50 µl of ABC reaction buffer (50 mM Tris, 50 mM KCl, 10 mM MgCl(2), 2 mM ATP, 5 mM dithiothreitol, and 50 µg/ml BSA, pH 7.4). Where indicated, 200 ng of pBR322 and TRCF construct (400 nM) were added, and incubation was at 37 °C for 25 min. Bound and free DNA were separated with a 5% polyacrylamide gel run at room temperature in TBE as described in Reardon et al. (1993).


RESULTS

An mfd Null Mutant

The first mfd mutation was obtained by Witkin in E. coli B/r (Witkin, 1966). This mutation, called mfd-1 shows the following phenotypic properties in B/r and/or K-12 backgrounds: (i) lack of mutation frequency decline following mutagenesis by UV and alkylating agents and holding in media that lacks amino acids; (ii) an about 3-fold increase in induced mutation rate; and (iii) less than a 2-fold increase in sensitivity to UV irradiation (Witkin, 1966; Oller et al., 1992; Selby and Sancar, 1994). The nature of the mfd-1 mutation is not known. It was conceivable that mfd-1 was a missense mutation and that mfd was an essential gene because the transcription-repair coupling factor performed another essential function. In fact the level of Mfd protein (about 500 per cell) is considerably higher than the levels of all three Uvr proteins which are required for transcription-repair coupling (see Selby and Sancar (1994)). To test whether or not Mfd was an essential protein we made a deletion mutant by replacing about 80% of mfd with a kanamycin-resistance encoding gene cassette. The resulting strain, UNCNOMFD (Deltamfd) behaved identically to the mfd-1 mutant derivative of AB1157 in all aspects, including slightly increased sensitivity to UV irradiation and lack of transcription-repair coupling in vitro (data not shown). Thus, we conclude that, whether or not Mfd performs a function separate from transcription-repair coupling, it has no essential cellular role.

Structural Domains of TRCF

The sequence of Mfd protein revealed several structural motifs: a 140-amino acid region of homology to UvrB within the amino-terminal third, a ``helicase domain'' within the carboxyl-terminal half, and a potential leucine zipper motif near the carboxyl terminus, as shown schematically in Fig. 1. These sequence motifs were assigned certain structural/functional duties in carrying out the TRC such as binding to UvrA, RNA Pol, or DNA. To test these predictions we made a deletion mutant and 5 fusion constructs carrying various regions of the protein shown schematically in Fig. 1, and a point mutant where the lysine residue in the Walker A sequence (``helicase motif IA'') was replaced by glutamine.

Binding of TRCF to Nucleic Acids

The binding was tested by footprinting and gel retardation. Fig. 3shows that wild-type TRCF does not form a stable complex with DNA in the absence or presence of ATP but a stable complex is formed when ATPS is included in the reaction mixture without or with ATP. In contrast, the GNT (K634N) mutant which has drastically diminished ATPase activity binds to DNA in the presence of ATP (lane 8) or ATPS (lanes 9 and 10). With the poorly hydrolyzed ATPS the resulting complexes are long-lived and thus can be more clearly detected by the gel retardation assay. We conclude that the TRCFbulletATP complex binds to DNA and that hydrolysis of ATP dissociates the protein-DNA complex. This finding parallels the involvement of ATP hydrolysis in the dissociation of a stalled RNA Pol by TRCF. The DNase I footprinting experiment in Fig. 4shows that RNA Pol forms a stable, stalled elongation complex at the site of a psoralen monoadduct in the template strand, both in the presence and absence of ATPS (lanes 1 and 2, all reactions contained ATP). As demonstrated previously (Selby and Sancar, 1993), TRCF dissociates the stalled RNA Pol in the absence of ATPS (lane 3). Dissociation is inhibited approximately 50% by ATPS (lane 4). Since both of these events, that is, the dissociation of TRCF from DNA and the dissociation of a stalled RNAP require hydrolysis of ATP, it is likely that they both result from the same process.


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 ATPS 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.



Footprint of TRCF

The nature of the DNA-protein complexes formed with the TRCF and double-stranded DNA was probed by DNase I footprinting using fragments of 184-2100 bp in size. A 3`-terminally labeled 2100-bp fragment was digested with various restriction enzymes to generate a series of shorter fragments, all of which had the same 3`-end labeled terminus. When the longest fragment was digested with DNase I in the presence of t-TRCF, it revealed alternating protected and hypersensitive regions (Fig. 6, lanes 1 and 2). Since this mode of DNase I digestion is consistent with the DNA being wrapped around the protein we wished to know whether there was a ``phasing'' sequence which initiated the wrapping of DNA at a preferred site or if the DNA wrapped around TRCF starting at any contact point. When footprinting was conducted with the shorter fragments the regions of protection relative to the labeled end remained the same. As an example, the region 330-360 nucleotides from the labeled terminus is protected in lanes 2, 4, 6, and 8 (Fig. 6) even though the distances between this region and the unlabeled terminus are about 1750, 630, 290, and 130 bp, respectively. The data are consistent with a phasing sequence (or structure) but the available data does not allow us to state what sequences constitute preferential contact sites with TRCF. We note that the tac promoter is present on all of these fragments except the 184-bp fragment.


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).



DNA Binding Domain of TRCF

By using a deletion mutant (t-TRCF), NH(2)- and COOH-terminal fusion constructs, and gel retardation assay in the absence or presence of ATPS (Fig. 7), the DNA binding domain was narrowed down to the region encompassing amino acids 571-938 (lanes 2-4 and 6-9). Again the binding of the mutant proteins (except GNT which binds to but poorly hydrolyzes ATP) was detectable only in the presence of ATPS. Thus, we conclude that the binding of ATP to the helicase domain of TRCF enables the protein to bind DNA through contacts made by this domain.


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 ATPS; in the bottom panel (B), ATPS 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 ATPS only the ATPase active site mutant retards the DNA, and even in this case retardation is not as pronounced as in the presence of ATPS because of the residual ATP hydrolysis by the mutant (lane 5).



Binding to RNA Polymerase

RNA polymerase does not bind to a TRCF affinity column under conditions where UvrA binds almost quantitatively (Selby and Sancar, 1993; data not shown); yet, TRCF does displace RNA Pol stalled at a lesion. Thus, it must interact with RNA polymerase in a stalled elongation complex. Since it does not specifically interact with damaged DNA or the other nucleic acid structures inherent to the stalled elongation complex, we made further attempts to detect TRCF-RNAP binding using procedures with greater sensitivity. We employed the pull-down assay in which TRCF-MBP fusion protein stably linked to amylose resin is incubated with RNA Pol, and after washing the unbound (Free, F) proteins from the resin, the bound (B) proteins are eluted with high salt and examined upon silver staining of SDS-polyacrylamide gels. Fig. 8A shows the result of an experiment in which RNA Pol was incubated with 50 µl of amylose resin linked to 98 pmol of either MBP or full-length TRCF-MBP fusion protein. UvrA was used in separate reactions as a positive control. The results clearly show that both UvrA and RNA Pol bind to TRCF, and RNA Pol binds less efficiently. A low level of subunit in the bound fraction indicates that TRCF is able to bind holoenzyme. The alpha and beta subunits are clearly seen in the bound fraction; however, beta` comigrates with a low level of TRCF-MBP which is present in the bound fraction due to contamination of the fraction with resin. Binding of beta` not only can be assumed based on the very strong association of the core subunits, but is also demonstrated in Fig. 8B, which shows the degree of binding of RNA Pol to the different TRCF-MBP fusion constructs that could be obtained at high concentrations (98 pmol/50 µl of resin). Interestingly, there is no binding to the C-P construct (lanes 3 and 4) which contains the potential leucine zipper motif. This region was considered a potential site of contact with the beta subunit of RNA Pol, since beta also possesses a potential leucine zipper motif. In contrast, the other two partial TRCF-MBP fusion constructs, which possess either the carboxyl-terminal two-thirds of TRCF (lanes 7 and 8) or the amino-terminal one-half of TRCF (lanes 5 and 6), do bind RNA Pol. These findings suggest that amino acids 379-571 are involved in binding RNAP; other regions of TRCF may also be involved but the carboxyl-terminal 22% of TRCF does not bind RNA Pol by itself.


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 beta`. 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.

UvrA Binding Domains of TRCF

Amino acids 82-219 of TRCF show 22% sequence identity with the corresponding region of UvrB protein (Selby and Sancar, 1993). Since both TRCF and UvrB bind to UvrA protein (see Selby and Sancar, 1994) it was suggested that this region of sequence identity might be involved in binding to UvrA. This prediction was tested by using a pull-down assay to examine binding of UvrA to the different regions of TRCF shown in Fig. 1. The results in Fig. 10show that under the conditions used, UvrA did not bind to constructs encompassing the carboxyl-terminal two-thirds of TRCF (lanes 3-8). However, binding to the amino-terminal constructs was detected (lanes 9-12) and the binding region was localized to the amino-terminal one-third of TRCF (lanes 9 and 10), which encompasses the region of sequence similarity with UvrB. Thus, these results are in agreement with the prediction that the UvrB homology region is involved in binding to UvrA.


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^Q (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.




DISCUSSION

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 UvrA(2)bulletUvrB(1) 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(2) UvrB(1) 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.



DNA Binding

The TRCF contains the so-called helicase motifs and thus this region of the protein was expected to interact with DNA. This expectation has been confirmed. Deletion mutants carrying the helicase domain bind DNA and those missing the entire region such as N-V (which contains residues 1-571) failed to bind DNA. Unexpectedly, we also found that ATP binding was required for DNA binding and ATP hydrolysis was necessary for dissociation of DNA-protein complexes. This is in contrast to other DNA binding ATPases such as UvrA, which bind to DNA with high affinity in the absence of ATP and dissociate from DNA upon ATP hydrolysis (Reardon et al., 1993). The unique action mechanism of TRCF suggested that it may have a specific affinity to DNA structures similar to the ``transcription bubble.'' However, actual binding experiments revealed no preference of TRCF for a duplex with a 12-nucleotide long bubble compared to duplex DNA and no preference for RNA-DNA hybrids, either in a duplex or in a three-strand intermediate. Most significantly the TRCF had no measurable affinity for RNA which strongly suggests that its reaction mechanism is radically different from the rho termination factor which also has helicase motifs and which also dissociates an RNA PolbulletRNAbulletDNA ternary complex. Finally, the DNA binding mode of TRCF appears to involve wrapping of the DNA around the protein.

RNA Pol Binding

The reaction catalyzed by TRCF, that is coupling of repair to transcription, implies specific interaction between TRCF and RNA Pol. Support for this prediction was obtained from an experiment which showed that TRCF displaced a stalled E. coli RNA Pol from a lesion site but did not displace a stalled T7 RNA Pol (Selby and Sancar, 1993). The failure of TRCF to preferentially interact with the nucleic acid components that are present in the stalled elongation complex also implies that a TRCF-RNA Pol interaction occurs. Direct evidence of this interaction was obtained using the pull-down assay. The interaction was found to occur in the absence of ATP. Furthermore, results of the pull-down and gel shift assays indicate that residues 379-571 are involved in the TRCF-RNAP interaction. Residues 1-378 are not required since the C-X fusion protein (residues 379-1148) was capable of removing the stalled RNAP. Our results do not rule out the possibility that residues 571-895 of the helicase motifs directly interact with RNA Pol; however, this region is already known to interact with DNA and ATP. Since ATP hydrolysis is required for both the dissociation of TRCF from DNA and removal of stalled RNA Pol, it seems likely that the helicase motifs participate in removing stalled RNAP as a consequence of their interaction with DNA and ATP while residues 379-571 are bound to RNA Pol. Residues 896-1148 of the carboxyl terminus did not stably bind RNA Pol but these residues also appear to be involved in dissociating the ternary complex, since the t-TRCF (residues 1-938) bound but did not remove the stalled RNA Pol. Thus, the predicted interaction of TRCF and RNA Pol through a leucine zipper structure in the carboxyl terminus (Selby and Sancar, 1993) appears unlikely. While the sequence of this region has several characteristics of a leucine zipper, it also has 2 proline residues which are not common to this structure. Furthermore, sequence analysis of the TRCF from Bacillus subtilis(^2)has not revealed any potential leucine zipper.

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.

UvrA Binding

The prediction that an NH(2)-terminal TRCF region of 140 amino acids with high sequence identity to a similar region in UvrB is a UvrA binding site has held up. Indeed this region binds quite tightly to UvrA and the carboxyl-terminal two-thirds of TRCF has no affinity to UvrA at all. Along these lines, it must be noted that a similar situation has been found with UvrB: the TRCF homology region at the NH(2)-terminal half of UvrB also binds with high affinity to UvrA. In addition, the carboxyl-terminal one-third of UvrB also interacts with UvrA with high affinity. (^3)The presence in UvrB of two separable UvrA binding sites, one of which presumably binds to the same region of UvrA that TRCF binds, probably is important in the transcription-repair coupling reaction.


FOOTNOTES

*
This work was supported by the National Institutes of Health Grant GM32833, a grant from the Human Frontier Science Program, and Grant CTR 3852 from the Council for Tobacco Research-USA, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: TRCF, transcription-repair coupling factor; Mfd, mutation frequency decline; TRC, transcription-repair coupling; MBP, maltose-binding protein; bp, base pair(s); kbp, kilobase pair(s); Pol, polymerase; BSA, bovine serum albumin; ATPS, adenosine 5`-O-(thio-triphosphate); ss, single stranded.

(^2)
Dr. J. C. Alonso, personal communication.

(^3)
D. S. Hsu, Q. Sun, and A. Sancar, unpublished observation.


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