ABSTRACT
One form of nucleotide excision repair (NER) is known to be functionally coupled to transcription, but the nature of this functional link in Escherichia coli is still unclear. Here we have employed the isolated membrane-associated nucleoids from E.coli to examine this issue. We show that the isolated nucleoid fraction is capable of excision of UV-induced pyrimidine dimers when reconstituted with a cytoplasmic fraction resolved by sucrose gradient fractionation. This excision activity by UvrABC is sensitive to rifampicin and is dependent on transcription. By using crosslinking and immunoprecipitation, the damage recognition protein, UvrA, was found to be specifically associated with the RNA polymerase [beta] subunit on the chromosomal DNA independent of DNA damage. It suggests that at least in one of the NER pathways the search for damage may be directly linked to RNA polymerase. In addition, the role of transcription in the unfolding of the nucleoid structure to allow repair enzymes to gain access to the damaged DNA is described. This study provides insight into the understanding of the transcription-repair coupling in vivo.
Nucleotide excision repair (NER), in monitoring the integrity of DNA, is responsible for removing a large variety of damaged nucleotides as oligomers. In Escherichia coli, the UvrABC endonuclease is responsible for this repair pathway. UvrA is a damage recognition protein; in vitro, it forms a UvrA2B complex with UvrB and delivers UvrB to a lesion on the DNA. UvrC then binds to UvrB bound-damaged DNA to induce dual incisions (1,2). In vivo, this repair pathway functionally cooperates with other cellular processes such as transcription. Such a functional link was first observed in mammalian cells by Bohr et al. (3). They discovered that actively transcribed dhfr genes of Chinese hamster ovary cells were repaired faster than total genomic DNA. This observation was extended by Mellon et al., (4) to show that the repair was significantly faster in the transcribed strand than in the non-transcribed strand. These findings were further observed both in the yeast Saccharomyces cerevisiae (5) and in E.coli (6).
Preferential repair implies some means of communication that must exist between Uvr proteins and RNA polymerase. Selby and Sancar discovered the transcription-repair coupling factor (TRCF) encoded by the mfd gene (mutation frequency decline) (7, for review MFD see reference 8). In mfd- cells, the repair of transcribed strands is reduced and, as a consequence, mutations are biased toward this strand (9). In a defined in vitro system, the purified Mfd protein can displace RNA polymerase stalled at a lesion; it can interact with UvrA and can stimulate the rate of repair of the transcribed strand during transcription (10). In the proposed model, Mfd protein releases the RNA polymerase blocked at a lesion and then escorts the repair proteins to the lesion site. However, the mfd- cells are not particularly UV sensitive (11). The latter observation suggests that the contribution of Mfd protein to strand specific repair for UV survival is not a major one. Hence, the nature of the functional link between repair and transcription in E.coli is still unclear.
To understand the nature of NER in E.coli, we attempted to isolate functionally active repair complexes and to characterize their structural and functional properties. We previously showed that Uvr proteins and DNA damaged sites are relocated to the inner membrane following UV irradiation of the cells (12). Further characterization of the isolated membrane-associated nucleoids reveals that a number of NER proteins together with proteins from the transcription machinery are recruited to DNA-membrane contacts. In the present study, we employed the isolated membrane-associated nucleoids to demonstrate the nature of transcription-repair coupling.
The following E.coli strains were used: wild-type strain MH1 [araD139, [Delta](ara,leu)7697, [Delta]lacX74, galU, galK, StrA]; uvrA deletion mutant strain MH1 [Delta]A [araD139, [Delta](ara,leu)7697, [Delta]lacX74, galU, galK, StrA, [Delta]uvrA]; uvrB deletion mutant strain N364 [w3110 gal+, sup°, F-, [Delta](att-bio-uvrB)]; Rifs strain MG1655 [btuB::Tn10 rpoB+]; Rifr mutant CAG3315 [btuB::Tn10 rpoB8 (Q513P)].
Escherichia coli cells were inoculated to an initial OD600 = 0.05 from overnight cultures and grown at 30°C in M9 medium. To label chromosomal DNA, 2 µg/ml thymine and 10 µCi/ml [3H]thymidine (Amersham, 48 Ci/mmol) were added to the culture initially. To label proteins, 10 µCi/ml [35S]sulfuric acid (NEN) was added to the culture 40 min before the cells were harvested. Cells were harvested at OD600 = 0.5-1.0 by centrifugation and resuspended in cold 0.01 M MgSO4 (OD600 = 0.1). Cell suspensions (25 ml) were UV-irradiated (254 nm) by the germicidal lamp (15 W) in a 14 cm diameter Petri dish. Cells were UV-irradiated at 10 or 40 J/m2 measured with a UV intensity meter (Hoefer).
The membrane-associated nucleoids were isolated as previously described (12).
Escherichia coli cells were labeled with [3H]thymidine and UV-irradiated at 40 J/m2. Irradiated cells were lysed and fractionated into the nucleoid fraction (DM fraction) and soluble fraction (`TOP' fraction) by sucrose gradient fractionation as previously described (12). The DM and `TOP' fractions were mixed and supplemented with final concentrations of 1 mM ATP, 100 µM each of UTP, CTP, GTP and 10 µM each of dNTPs. The mixtures were incubated at 37°C. At selected times, equivalent to 109 cells of mixture were removed to measure percentage of thymine dimer remaining in genomic DNA using the method of Cook and Friedberg (13).
The transcription activity was measured by incorporation of [3H]uridine into acid-insoluble material according to the method of Swenson and Setlow (14). Cells were pretreated with rifampicin for 10 min before UV irradiation (40 J/m2). Irradiated cells were collected and resuspended in original M9 culture supplemented with 1 µCi/ml 5,6-[3H]uridine (Amersham, 34 Ci/mmol) to continue incubation. At selected times, a 0.1 ml sample of labelled cell suspension was added to 1.0 ml of cold 10% trichloroacetic acid. After 30 min incubation on ice, the trichloroacetic acid-precipitated material was collected on HA-type filters (Millipore), washed three times with 5 ml of cold 5% trichloroacetic acid followed by 5 ml of absolute ethanol. The filters were dried and then subjected to scintillation counting.
Isolated membrane-associated nucleoids (DM fraction) were treated with increasing doses (0-2 mM) of crosslinker dithiobis-(succinimidylpropionate), DSP (Pierce, 20 mM stock solution in dimethyl formamide). The crosslinking reaction was carried out on ice for 20 min. The reaction was then quenched by the addition of 1 M Tris-HCl, pH 7.5, to a final concentration of 50 mM. The nucleoids were further treated with DNase I to isolate the D (DNA-bound proteins) fraction and the M (membrane proteins) fraction as previously described (12). Samples were then subjected to electrophoresis for western blot analysis or to immunoprecipitation.
Escherichia coli cellular proteins were 35S-labelled. The membrane-associated nucleoids were isolated, treated with DSP and then fractionated into D and M fractions as described above. Samples (200 µl) of the crosslinked products from the D fraction (in sucrose gradient buffer) were added with 2 µl of anti-UvrA polyclonal antibody, BSA to 1%, and NaCl to 150 mM and incubated for 2 h at 37°C. Subsequently, 50 µl of anti-rabbit Ig immunobead (BioRad) was added to the mixture and incubated for 3 h at 37°C with gentle rocking. The antigen-antibody complexes were collected by centrifugation at 10 000 g for 20 s and then resuspended in 1 ml of wash buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA and 150 mM NaCl). The resuspended beads were incubated for 20 min with rocking. The washing procedure was repeated four times. The final washed immunobeads were collected, resuspended in 1× SDS (sodium dodecyl sulfate) gel-loading buffer, and subjected to electrophoresis.
A procedure for the isolation of membrane-associated nucleoids from E.coli was followed as previously described (12). The E.coli spheroplasts were carefully lysed by the non-ionic detergent {3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate}, CHAPS, in the presence of spermidine. This treatment preserves the membrane-attachment sites associated with the folded chromosomal DNA. Sedimentation of these lysates in a sucrose gradient containing Mg2+ yields a single peak of membrane-associated nucleoids, DNA-membrane complexes (DM fraction). The remaining proteins present in the applied lysate (`TOP' fraction) include cytoplasmic proteins and CHAPS-soluble inner membrane proteins. Spermidine and Mg2+ provide counterions to maintain chromosomal integrity as well as the reduction of aggregation at high chromosome concentrations. Of the chromosomal DNA from the lysate >95% was recovered at 2 × 1010 chromosomes per ml. This chromosome suspension is relatively shear-resistant, allowing for ease of manipulation. Characterization of repair protein distribution showed that UvrB is present exclusively in the `TOP' fraction; most of the other proteins involved in NER are associated with DNA-membrane complexes (12).
Whether the isolated membrane-associated nucleoid (DM fraction) is competent to carry out excision repair when reconstituted with the `TOP' fraction was examined. Wild-type E.coli cells were incubated with [3H]thymidine to label chromosomal DNA, and UV-irradiated at 40 J/m2, generating ~1-2 × 103 thymine dimers per genome. The cells were immediately lysed and resolved into the DM and `TOP' fractions. These two fractions were mixed, supplemented with all four ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs), and incubated at 37°C for evaluating repair by measuring the removal of UV-induced thymine-containing pyrimidine dimers from the genomic DNA (13). As shown in Figure 1, ~80% of the thymine dimers were removed in vitro within 10 min after mixing the DM fraction with `factors' in the `TOP' fraction. Dimer removal was not observed in the DM fraction alone. The remaining 20% of the thymine dimers were not removed either by extending the incubation time or supplementation with excess `TOP' fraction proteins, rNTPs or dNTPs. This excision activity was not an artifact resulting from DNA degradation by the factors in the `TOP' fraction because no dimer removal was detected when DM fraction from wild-type cells was reconstituted with `TOP' fraction from [Delta]uvrB mutants (Table 1, experiment 5; described below). By comparison with the in vivo kinetics of dimer removal (12), this nucleotide excision activity appears to represent the early stages of repair catalyzed by constitutive repair enzymes. The removal of the residual 20% of the dimers which requires SOS-induced de novo protein synthesis (12) was not evident in this reconstitution system.
Reconstitution permits the analysis of those proteins and cofactors associated with the repair of intact chromosomal DNA. These results are shown in Table 1. The specific excision activity is thermolabile (experiments 3-4) and is UvrABC-dependent because NER activity is not detected by the reconstitution of DM from wild-type cells with `TOP' from [Delta]uvrB mutants (experiment 5).
The `TOP' fraction contains endogenous rNTPs and dNTPs since the dialysed `TOP' fraction requires supplementation with both rNTPs and dNTPs to restore the excision activity (described below). In the absence of supplementation with the four exogenous rNTPs, there is only ~40% of excision repair activity (experiment 6). The effect of the addition of ATP becomes apparent in causing an increase in activity to 87% (experiments 7-8). The optimal level of dimer removal is achieved only with the addition of all four rNTPs (experiment 9), which suggests the participation of concomitant transcription in the excision of pyrimidine dimers from chromosomal DNA. Experiment 9 also indicates that dNTPs are not limiting for this complemented system because supplementation is not required for restoration of full activity. To examine the role of resynthesis in the excision process, the `TOP' fraction was dialyzed to remove the endogenous rNTPs and dNTPs. The complemented system was then supplemented with these components. Experiments 10-13 reveal that addition of only the four rNTPs is not sufficient to generate excision activity. The addition of both dNTPs and rNTPs, however, restores 100% of excision activity. These results indicate that both transcription and the resynthesis are required for the excision process.
Table 1.
We previously showed that most of UvrA, UvrD, DNA polymerase I and 40% of the total UvrC are located in the DM fraction and that UvrB is present in the `TOP' fraction (12). These proteins are sufficient for in vitro excision of dimers in UV-damaged plasmid DNA. However, in experiments 14-16, complementation of the DM fractions with purified UvrB or UvrC is not sufficient for excision repair activity. These results suggest that other `factors' in the `TOP' fraction, besides Uvr proteins, are required for dimer excision in intact folded chromosomal DNA. Furthermore, this excision activity does not require nascent proteins because it is insensitive to chloramphenicol (experiments 17-21), suggesting that it is a constitutive excision activity.
The linkage of NER to transcription becomes more apparent in that excision of thymine dimers requires the four rNTPs (Table 1, experiments 6-9). To further confirm these results, wild-type cells were treated with rifampicin to inhibit transcription before isolation of the nucleoids. Experiments 22-25 show that the excision activity of the isolated nucleoids is sensitive to the rifampicin treatment. Although rifampicin is a specific inhibitor of transcriptional events, its specificity in this system was confirmed by the use of rifampicin resistant strains (Rifr) of E.coli. The particular Rifr mutant used in this study is a point mutation located in the center of the rpoB ([beta] subunit) gene (15). Such a mutation leads to resistance to extremely high concentrations of rifampicin in vivo (>1000 mg/ml). Experiments 26-27 show that rifampicin, even at very high concentrations (10 µg/ml), had no effect on excision activity in the reconstitutions isolated from Rifr mutants. Furthermore, reconstitution of the DM fraction from Rifr and the `TOP' fraction from Rifs results in only 13% decrease of excision activity by rifampicin, while rifampicin significantly inhibited the excision activity when reconstituted the DM fraction from Rifs and the `TOP' fraction from Rifr (experiments 28-29). These results suggest that (i) the functioning RNA polymerase is important in excision repair in the reconstituted system, and (ii) RNA polymerase is not the missing `factor' in the `TOP' fraction.
The sensitivity of the excision activity to rifampicin was further examined in intact cells. Rifr and Rifs cells were pretreated with increasing doses of rifampicin for 10 min before UV irradiation. The influence of rifampicin in excision repair and transcription activities were then determined following irradiation. As shown in Figure 2, not only transcription but also excision activities were sensitive to rifampicin treatment in Rifs cells, but not in Rifr cells. This effect was not due to a decrease of expression in Uvr proteins by rifampicin because western blotting data showed that steady state levels of the Uvr protein in Rifs following rifampicin treatment were not significantly decreased in the first 30 min post-UV (data not shown). This effect was also not due to inhibition of Uvr proteins by rifampicin because rifampicin had no effect on the excision repair of UV-damaged plasmid DNA by purified Uvr proteins (data not shown). These results suggest that excision of UV-induced pyrimidine dimers in E.coli is dependent on transcription.
That excision repair is dependent on transcription suggests that repair proteins may be physically associated with RNA polymerase and that repair enzymes are accessible to DNA only during transcription, resulting in loosening of the nucleoid structure. The possibility of physical interaction between Uvr proteins and RNA polymerase subunits was studied by using reversible crosslinking. Isolated E.coli nucleoids were treated with increasing doses of the crosslinker DSP immediately after fractionation. DSP, with a 12 Å arm-length, reacts with primary amine groups and the crosslink reaction is easily reversed by reduction (16). DSP-treated nucleoids were digested by DNase I to release the specific DNA-bound proteins. After centrifugation, the membranes were pelleted. Such pellets contain the DNA-membrane contact proteins and outer membrane proteins (M fraction) and the supernatant fraction contains the DNA-bound proteins (D fraction) as previously described (12). The crosslinked products in the D fraction were resolved by SDS-PAGE and identified by western blotting. As shown in Figure 3, in the wild-type cells, the electrophoretic mobility of one of the UvrA-containing crosslinked products was the same as that of one of the RNA polymerase [beta] subunit containing crosslinked products (lanes 1-4 and 6-9), but this specific crosslinked product was not found in [Delta]uvrA mutants (lane 11). Moreover, this crosslinked product was found not only in UV-irradiated nucleoids but also in those that were not irradiated (lanes 4-5 and 9-10). To further confirm these results, immunoprecipitation was performed. The cellular proteins of wild-type E.coli cells and [Delta]uvrA mutants were 35S-labeled. The nucleoids were isolated, incubated with or without DSP, and further fractionated into the D and M fractions. The proteins in the D fraction were immunoprecipitated with anti-UvrA polyclonal antibodies and analyzed by SDS-PAGE under reducing conditions. As shown in Figure 4, in the absence of DSP treatment, no RNA polymerase [beta] subunit was co-immunoprecipitated with UvrA (wild-type, DSP 0 mM). However, the RNA polymerase [beta] subunit was co-immunoprecipitated with UvrA in a concentration-dependent manner with DSP (wild-type, DSP 0.5-1.0 mM). Importantly, in [Delta]uvrA mutants, as a negative control, RNA polymerase [beta] subunit was not immunoprecipitated, indicating that (i) the anti-UvrA antibodies were not crossreacted with the RNA polymerase [beta] subunit and (ii) it rules out a non-specific artifact, such as RNA polymerase [beta] subunit crosslinked to other proteins and non-specifically immunoprecipitated by the antibodies.
The role of transcription in unfolding of the nucleoid structure to allow repair enzymes to gain access to the damaged DNA was examined. The physical structure of the nucleoid under different physiological conditions is reflected in its accessibility to nucleases. It is suggested that the more exposed the nucleoid structure is to nucleases the more likely it will be accessible to repair enzymes. In this study, DNase I was used to measure such structural changes. Reaction conditions were first standardized by treating the isolated nucleoids with increasing levels of DNase I to determine the sensitivity of the nucleoid to digestion. The identical concentration of nucleoids, when isolated under different physiological conditions, were treated with DNase I under the same reaction conditions. As shown in Figure 5A, the nucleoids are much less sensitive to DNase I when isolated from cells that were treated with rifampicin (+rif, -UV) compared to those without treatment (-rif, -UV), suggesting a possible role for transcription in providing access of the nucleoid structure to repair proteins. In addition, the nucleoid is more sensitive to DNase I when isolated from cells undergoing rapid repair of UV-damaged chromosomal DNA (-rif, +UV) compared to those without UV (-rif, -UV), suggesting that during repair the nucleoid has a more accessible structure. These results are consistent with our previous observations that during repair the chromosomal structure appears to be more dispersed (12).
In the present study we have employed isolated membrane-associated nucleoids to study the functional linkage between transcription and NER.The protocol for the isolation of membrane-associated nucleoids was designed to incorporate the methods established and characterized by Worcel and co-workers. They examined the structure of isolated nucleoids by electron microscopy where they demonstrated membrane patches that anchor to DNA (17). These isolated nucleoids were able to replicate their DNA when supplemented with soluble enzyme fractions (18). In this study we observed that nucleoids isolated by the Worcel method also has NER capacity when reconstituted with cytoplasmic protein fractions. Two factors are important for the isolated nucleoids to carry out NER in vitro. First, the compact state of the folded chromosome seems to influence its excision capacity. When nucleoids are isolated in limiting spermidine concentrations (<10 mM), there is an increase in viscosity and decrease in excision capacity. Secondly, membrane association with the nucleoid seems to be important for its excision capacity. When nucleoids are isolated in elevated CHAPS concentrations (>14 mM), there is a corresponding decrease in membrane content and excision capacity. These and other data (12) correlate with the data of Worcel and his co-workers (17,18), which suggests that nucleoids isolated in these experiments were of comparable quality to those obtained by Worcel's group.
The results reported here demonstrate that E.coli NER activity, at least in a reconstituted system, is dependent on concomitant transcription. This is suggested from the required supplementation of the dialyzed cytosolic fraction with the four rNTPs (Table 1) and the sensitivity of the excision activity to rifampicin in vitro (Table 1) and in vivo (Fig. 2). By using reversible crosslinking and immunoprecipitation, we showed that the UvrA protein is able to specifically crosslink to the RNA polymerase [beta] subunit in the chromosomal DNA fraction and this association is DNA damage independent (Fig. 3). These results suggest that some of the UvrA protein is weakly associated with or physically close to RNA polymerase during transcription. The transcription-repair complex can interact in the following two ways: firstly, the UvrA protein may directly interact with RNA polymerase as a lesion sensor during transcription. When a DNA lesion is encountered, UvrB may cooperate with UvrA to unwind the template when driven by the UvrA2B-associated helicase activity. This may eventuate in the signaling of a DNA-damage site, directing the arrival of a set of `repair factors', which could displace RNA polymerase and repair the damaged site. Secondly, it has been demonstrated that UvrA2B has a 5'-3' helicase activity (19), which may allow the UvrA2B to translocate along DNA in search of damaged sites (20). It has also been shown that when RNA polymerase binds to its promoter site it generates a `bubble' providing for a `landing site' to which the UvrA2B can bind (21,22). Thus, UvrA2B may associate with the RNA polymerase at its promoter site during initiation and then can translocate on the non-transcribed strand when coupled with RNA polymerase during elongation.
The association of RNA polymerase and repair enzymes seen in E.coli is observed similarly in higher organisms. The human transcription factor BTF2/TFIIH and its yeast counterpart, were found to contain a component originally characterized as NER enzymes, including ERCC3 (SSL2/Rad25), ERCC2 (Rad3) and p44(SSL1) (23-25). TFIIH possesses a Uvr A2B-like helicase activity, probably coupled to the ERCC3 and ERCC2 subunits, which may be required for transcription coupled to repair.
We also demonstrated the potential role of transcription in accessing the nucleoid structure to repair proteins when measured by DNase I sensitivity (Fig. 5). The DNA packing density of the E.coli nucleoid should, theoretically, allow enzymes or even transcription events to penetrate into a viscous sol (26). Nevertheless, from immunolabeling experiments it has been shown that the transcriptional activity of the nucleoid is not associated with its bulk but rather is restricted to its surface according to the presence of nascent RNA, RNA polymerase and ribosomes (27-29). Furthermore, the majority of UvrA molecules are localized on the surface of the nucleoid, not in the bulk DNA where most of the damaged sites are localized (12). The central part of the inactive bulk DNA seems to be restricted from the active penetration of enzymes. Hence, those genes to be transcribed may have to reach the surface of the nucleoid. Perhaps there is continuous movement of DNA within the nucleoid as originally suggested by Ryter and Chang (27). Such movement of DNA may be one of the transcription events. When cells are treated with rifampicin in order to inhibit transcription activity, the movement of DNA may also be halted and the entire nucleoid becomes structurally inaccessible and functionally inactive. Thus, repair enzymes may be unable to reach the DNA for repair purposes (Fig. 5B). However, when cells undergo rapid repair, the nucleoid may be dispersed allowing those repair proteins to gain access to the damaged sites. These conclusions were revealed from the immunoelectron microscopy studies (12) and increased sensitivity of the nucleoid to nucleases (Fig. 5A). We previously showed that Uvr proteins and DNA damaged sites are relocated to the inner membrane following UV irradiation of the cells (12). One of the purposes for association of damaged DNA to the inner membrane may be to open the transcribing DNA domain for repair proteins.
From these studies we speculate that two repair pathways could contribute to preferential repair in transcribed genes in E.coli. First, the search for damage may be directly linked to RNA polymerase, leading to the preferential repair of the actively transcribed strands. Linkage can be accomplished by either the direct interaction of the UvrA protein with the [beta] subunit of RNA polymerase as a lesion sensor during transcription, or through coupled translocation of UvrA2B and RNA polymerase. A lesion in the template strand, when recognized by UvrA, can potentially block RNA polymerase. The stalled ternary transcription complex (UvrA-RNAP-RNA-DNA) may then localize at the inner membrane. After damaged DNA associates with the membrane, RNA polymerase may back off the lesion site, perhaps through the functioning of the transcriptional repair coupling factor (TRCF) (10), allowing the repair protein to function. The DNA lesion is incised by UvrB and UvrC, excised by UvrD, and replaced with normal nucleotides by DNA polymerase and ligase. The association of damaged DNA to the membrane results in unfolding of the transcribing DNA domain, thereby allowing Uvr proteins to gain access to the damaged DNA. This second pathway may contribute to the repair of the non-transcribed strand.
We would like to thank M.H.Sayre, S.S.Krag and V.L.Culotta (Johns Hopkins University) for helpful discussions; J.C.Matanoski for critical review of this manuscript and D.J.Jin for the gift of invaluable strains. The financial support for these studies was provided by a Merit Award to L.G. from the National Institutes of Health (GM-22846).
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Bacterial strains
Bacterial growth, UV irradiation and radioactive labeling conditions
Isolation of membrane-associated nucleoids
Reconstitution experiment and dimer excision assay
Transcription activity assay
Crosslinking conditions
Immunoprecipitation conditions
Results
Reconstitution of `TOP' and DM fraction
Requirements for excision repair in reconstituted system
NER and transcription
Association of UvrA with RNA polymerase [beta] subunit
Accessibility of the nucleoid to Uvr proteins
Discussion
Acknowledgements
References
Reactantsa
Relative
removal of
[3H]thymine
dimers (%)b
1. DM (wt)c
0
2. DM (wt) + TOP (wt)
100
3. heated DM (wt)d + TOP (wt)
0
4. DM (wt) + heated TOP (wt)
0
5. DM (wt) + TOP ([Delta]uvrB )
0
6. DM (wt) + TOP (wt) - NTP
43 ± 5.2
7. DM (wt) + TOP (wt) - ATP
47 ± 6.1
8. DM (wt) + TOP (wt) - U(C,G)TP
87 ± 4.7
9. DM (wt) + TOP (wt) - dNTP
100
10. DM (wt) + dialysed TOP (wt)e
0
11. DM (wt) + dialysed TOP (wt) + NTP
0
12. DM (wt) + dialysed TOP (wt) + dNTP
0
13. DM (wt) + dialysed TOP (wt) + NTP + dNTP
100
14. DM (wt) + purified UvrBf
0
15. DM (wt) + TOP ([Delta]uvrB) + purified UvrB
100
16. DM (wt) + purified UvrB + purified UvrCf
0
17. DM (wt) + TOP (wt) + chloramphenicol
1 µg/ml
100
18. DM (wt) + TOP (wt) + chloramphenicol
10 µg/ml
100
19. DM (wt) + TOP (wt) + chloramphenicol
100 µg/ml
100
20. DM (wt) + TOP (wt) + chloramphenicol
300 µg/ml
100
21. DM (wt) + TOP (wt, -UV)
100
22. DM (wt) + TOP (wt) + rifampicing
0.04 µg/ml
52 ± 6.7
23. DM (wt) + TOP (wt) + rifampicing
0.2 µg/ml
23 ± 8.1
24. DM (wt) + TOP (wt) + rifampicing
1 µg/ml
0
25. DM (wt) + TOP (wt) + rifampicing
5 µg/ml
0
26. DM (Rifr) + TOP (Rifr) + rifampicin
5 µg/ml
100
27. DM (Rifr) + TOP (Rifr) + rifampicin
10 µg/ml
100
28. DM (Rifr) + TOP (Rifs) + rifampicin
5 µg/ml
87 ± 3.4
29. DM (Rifs) + TOP (Rifr) + rifampicin
5 µg/ml
37 ± 5.7
REFERENCES
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 27 Feb 1998
Copyright© Oxford University Press, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Q. Sun and W. Margolin Effects of Perturbing Nucleoid Structure on Nucleoid Occlusion-Mediated Toporegulation of FtsZ Ring Assembly J. Bacteriol., June 15, 2004; 186(12): 3951 - 3959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-B. Qiu, Y.-L. Lin, K. C. Thome, P. Pian, B. P. Schlegel, S. Weremowicz, J. D. Parvin, and A. Dutta An Eukaryotic RuvB-like Protein (RUVBL1) Essential for Growth J. Biol. Chem., October 23, 1998; 273(43): 27786 - 27793. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||