©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Function of Transcription-Repair Coupling Factor
II. CATALYTIC 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
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 A(2)B(1) 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.


INTRODUCTION

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) (^1)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 geq90 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.


MATERIALS AND METHODS

Strains

E. coli strains AB1157 (wild type with respect to repair), ES1484 (mutL218::Tn10), and ES1481 (mutS215::Tn10) were obtained from the E. coli Genetic Stock Center (Yale University).

Enzymes

The TRCF, its truncated derivative t-TRCF and deletion constructs fused to maltose-binding protein were constructed and purified as described previously (Selby and Sancar, 1995). The UvrA, UvrB, and UvrC proteins were purified as described by Thomas et al.(1985). RNA polymerase was purchased from Promega and Boehringer Mannheim Biochemicals. Rho was a kind gift of Dr. John P. Richardson (Indiana University). Helicase II (UvrD protein) was kindly provided by Dr. S. N. Matson (University of North Carolina). EcoRI (E111G) was kindly provided by Dr. J. Griffith (University of North Carolina). MutS protein was kindly provided by Dr. P. Modrich (Duke University).

Repair Assays

The pDR3274 plasmid used for transcription/repair assays has been described previously (Selby and Sancar, 1991, 1993). Both the repair synthesis assay (described in the accompanying paper; Selby and Sancar, 1995) and the nicking assays were used to measure repair. In the nicking assay the DNA is linearized, the template strand is terminally labeled, and the UV-irradiated DNA is then used in a transcription/repair reaction in which the repair is measured by the appearance of nicks in the irradiated DNA (Selby and Sancar, 1993).

Transcription Termination Reactions

Two types of templates were employed. To study the effect of TRCF on rho-dependent and rho-independent transcription terminations, a 1250-bp BstEII fragment carrying the lac UV5 promoter and the first 479 bp of lacZ was isolated from pMC1. This region of the lacZ transcriptional unit contains both rho-dependent and -independent transcription termination sites (Ruteshouser and Richardson, 1989). A single round transcription protocol was used. Reactions in either repair 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 bovine serum albumin) or glutamate buffer (40 mM Tris acetate, 6 mM magnesium acetate, 150 mM potassium glutamate, and 2 mM ATP, pH 7.8) were conducted in two steps. First, RNA Pol (0.03 unit, Boehringer Mannheim) and the TRCF derivative were incubated with the template at 37 °C for 10 min in 25 µl. Then 25 µl of repair or glutamate buffer with additions was added to bring GTP and UTP each to 270 µM, CTP to 20 µM, [P]CTP to 4 µCi per reaction and rifampicin to 22 µg/ml. Incubation continued at 37 °C for 20 min (unless otherwise indicated), and then samples were processed for analysis of RNA on 3.6% sequencing gels. pMC1 was generously provided by Dr. J. P. Richardson. An additional template used to examine rho-independent termination was the approximately 400-bp PvuII fragment purified from pGFIB-I (Normanly et al., 1986). This template bears a synthetic promoter upstream from the transcription termination sequences from the E. coli rrnC operon. pGFIB-I was generously provided by Dr. M. J. Rogers (Yale University).

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(2) to 4 mM, ATP to 1 mM, GTP and UTP to 200 µM each, CTP to 20 µM, [alpha-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.

Helicase Substrates

The substrates for DNA:DNA and DNA:RNA helicase assays were prepared by the method of Matson et al. (1983). The substrate for helicase activity on a RNA-DNA hybrid with a 5`-tail was prepared as described previously (Brennan et al., 1987). The RNA:DNA bubble duplex described in the accompanying paper (Selby and Sancar, 1995), was prepared as described by Daube and von Hippel(1992). The only difference was that the DNA strands were 90 nt in length. The ``transcription bubble'' generated by mismatch between the two strands was 12 nucleotides in length. The RNA was 20 nt with 12 nt hybridized to the ``transcribed'' strand and 8 nucleotides (all U) as the ``free'' non-hybridized 5`-tail (Daube and von Hippel, 1992). Helicase assays employed 30-min incubations of protein with substrate at 37 °C in repair buffer.


RESULTS

TRCF Lacks Helicase Activity

The TRCF has the so-called ``helicase motifs'' found in a number of DNA and RNA helicases (Gorbalenya and Koonin, 1993). However, our initial tests with DNA:DNA and 5`-tail-RNA:DNA substrates failed to reveal any such activity (Selby and Sancar, 1993). Considering the fact that in an elongation complex about 12 nucleotides of the transcript are hybridized with the template (Gamper and Hearst, 1982), and that TRCF releases both RNA Pol and the truncated RNA from a stalled elongation complex, it could be argued that TRCF does have a helicase activity that can be detected only with the appropriate substrate or with the appropriate concentrations of the protein. Therefore, we decided to investigate the helicase effect of TRCF in more detail.

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 A(2)B(1) 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 times are results of independent experiments with TRCF, triangles for HelII and squares for UvrA(2)B(1) 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, Delta, 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.

Effect of TRCF on Transcription Termination Sites

Both rho and TRCF release stalled RNA polymerase complexes although their requirements for interaction with such complexes might be different. To test for potential functional similarities between rho and TCRF we employed a DNA template with the lac UV5 promoter and rho-dependent and rho-independent transcription termination sites as well as ``natural'' pause sites for RNA Pol (Zou and Richardson, 1991). Fig. 2A shows that the TRCF does not affect transcription termination at either rho-dependent or rho-independent termination sites but it may affect termination at natural pause sites (see below). A curious effect that was observed in these experiments remains to be explained. In experiments with the template used in Fig. 2B, the presence of TRCF caused more than half of the transcripts to be over twice the size of the run-off transcript (Fig. 2B, lane 5) as though TRCF enables RNA Pol to switch templates at the end of the fragment and transcribe the complemetary strand. Of the various TRCF mutants tested two constructs, C-X (residues 379-1148) and C-V (residues 579-1148) appear to have an effect on transcription which is to cause premature termination in a fraction of the molecules (Fig. 2B, lanes 6 and 7). These results, taken together, suggest that TRCF interacts with elongation complex but most importantly reveal that TRCF has no role in physiological (non-damage dependent) transcription termination. These data also explain why rho null mutants are lethal (Richardson, 1993) but TRCF null mutants are not (Selby and Sancar, 1995).


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.



Effects of TRCF on RNA Polymerase Stalled at a Protein Roadblock

Repressors inhibit transcription by interfering with the binding of RNA Pol to the promoter and by blocking the progression of RNA Pol (see Pavco and Steege(1990)). In addition, genes may contain pseudosites for specific DNA binding proteins and these sites may be occupied at sufficiently high frequency to interfere with gene expression. Therefore, it is of interest to know whether RNA Pol is blocked by site-specifically bound proteins other than repressors and whether such blocks can be overcome by TRCF. Previously, it has been shown that an active site mutant of EcoRI (E111Q) when bound stably to an EcoRI site blocks transcription, and a stable elongation complex forms at the block site. When the E111Q is released by increasing the ionic strength, RNA Pol resumes transcription and elongates the truncated transcript (Pavco and Steege, 1990). However, if the rho protein is added to the stalled complex, it displaces RNA Pol and the truncated transcript from the road block (Pavco and Steege, 1990). When an analagous experiment was conducted it was found that TRCF functioned the same as rho factor (Fig. 3). The addition of TRCF to RNA Pol blocked by EcoRI (E111Q) releases most of the transcript in truncated form, presumably by releasing RNA Pol as well. Subsequent dissociation of EcoRI (E111Q) by high salt results only in a small fraction of full-length transcripts (compare lanes 2 and 4). Thus, we conclude that the TRCF is capable of dissociating stalled RNA Pol complexes blocked either by a lesion or by a specifically bound protein.


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).



Role of DNA Topology in Transcription-Repair Coupling

(A)BC excinuclease activity is generally unaffected by the topology of the damaged DNA substrate (see Selby and Sancar(1994)). In contrast, binding of RNA Pol to promoters and the rate of closed to open complex isomerization are effected by the superhelicity of the template (Mulligan et al., 1985). In view of this influence of topology on RNA Pol, we examined whether the topology of the template/substrate influenced transcription-coupled repair. The tac promoter-based transcriptional unit in pDR3274 is well suited for such experiments since the strength of this promoter is relatively unaffected by superhelicity (Mulligan et al., 1985). We compared the levels of transcription-repair coupling in UV-irradiated pDR3274 that was supercoiled, relaxed, or linearized. The results shown in Fig. 4confirm that repair of UV-damaged DNA is unaffected by superhelicity, and furthermore, the results clearly show that transcription-repair coupling also is unaffected by the DNA topology.


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^2 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.



Effect of Lesion Location on Transcription-Repair Coupling

The pDR3274 plasmid which contains the tac promoter was linearized with EcoRI which digests 235-bp upstream from the transcription start site. The template strand was 3` terminally labeled, the DNA was irradiated with UV and then the template/substrate was treated with (A)BC excinuclease, which incises at the fourth phosphodiester bond 3` and the eighth phosphodiester bond 5` to the photoproduct. Incision assay reaction products resolved on a 3.6% sequencing gel are shown in Fig. 5. In the absence of TRCF, the binary complex at the promoter, and the elongation complexes throughout the transcriptional unit inhibit repair of the template strand (compare lanes 1 and 2 in Fig. 5A, and lanes 7 and 8 in Fig. 5B). When TRCF is also included in the reaction mixture (lane 3 in Fig. 5A, and lane 9 in Fig. 5B) the repair in the area of the promoter to +11 is still inhibited. However, starting at the lesion at +15, the repair of all lesions downstream is stimulated, albeit to varying degrees. Thus, even though TRCF interacts with both binary and elongation complexes (Selby and Sancar, 1995), the coupling factor can only stimulate the excision of lesions blocking RNA Pol after it has entered its elongation mode.


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^2 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.

The Excision Repair Step Stimulated by the TRCF

In nucleotide excision repair the UvrB subunit is delivered to the damage site by the molecular matchmaking activity of the damage recognition subunit, UvrA; the two form an A(2)B(1) complex which binds to and locally denatures DNA. UvrA then dissociates, leaving the UvrB subunit bound to the lesion site (Orren and Sancar, 1989; Sancar and Hearst, 1993). The UvrB-DNA complex is then recognized by UvrC which upon binding induces UvrB to make the 3` incision which in turn enables UvrC to make the 5` incision (Lin and Sancar, 1992). A kinetic analysis has shown that formation of the UvrB-DNA complex is the rate-limiting step (Orren and Sancar, 1990). Therefore, it is predicted that the TRCF will enhance the rate of repair of the transcribed strand by enhancing the rate of this step.

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 A(2)B(1) 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(2)B(1)-damaged DNA preincision complex via reformation of the free A(2)B(1) 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(2)B(1) 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.



Role of MutL and MutS in Transcription-Repair Coupling

Strand-specific repair has been reconstituted in vitro in a completely defined system without the addition of MutL or MutS proteins (Selby and Sancar, 1993). However, recent in vivo experiments have found that strand-specific repair does not occur in UV-irradiated mutL and mutS cells, suggesting that there might be a direct involvement of these proteins in transcription-repair coupling. (^2)Therefore, we made cell-free extracts from both mutL and mutS mutant cells and tested them for coupling activity in vitro. Strand-specific repair was observed with both mutant extracts (Fig. 7). Significantly, when the mutS extract was supplemented with purified MutS protein the degree of strand-specific repair was unaffected (lanes 7 and 8). In contrast, supplementing the mutant extracts with TRCF enhanced the strand-specific repair of the CFE (lanes 4 and 9) as is commonly observed with wild-type extracts (Selby et al.(1991); see also Fig. 6, lanes 5 and 9). Thus, our in vitro data indicate that MutL and MutS are not directly involved in transcription-repair coupling.


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.




DISCUSSION

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 UvrA(2)bulletUvrB(1) 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.


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; bp, base pair(s); nt, nucleotide; Pol, polymerase.

(^2)
I. Mellon, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. R. Bockrath and I. Mellon for communicating their data prior to publication.


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