From the Sealy Center for Molecular Science,
University of Texas Medical Branch, Galveston, Texas 77555-1071 and
the § Department of Biological Sciences, Stanford
University, Stanford, California 94305
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ABSTRACT |
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UvrA is one of the key Escherichia coli proteins involved in removing DNA damage during the process of nucleotide excision repair. The relatively low concentrations (nanomolar) of the protein in the normal cells raise the potential questions about its stability in vivo under both normal and stress conditions. In vitro, UvrA at low concentrations is shown to be stabilized to heat inactivation by E. coli molecular chaperones DnaK or the combination of DnaK, DnaJ, and GrpE. These chaperone proteins allow sub-nanomolar concentrations of UvrA to load UvrB through >10 cycles of incision. Guanidine hydrochloride-denatured UvrA was reactivated by DnaK, DnaJ, and GrpE to as much as 50% of the native protein activity. Co-immunoprecipitation assays showed that DnaK bound denatured UvrA in the absence of ATP. UV survival studies of a DnaK-deficient strain indicated an 80-fold increased sensitivity to 100 J/m2 of ultraviolet light (254 nm) as compared with an isogenic wild-type strain. Global repair analysis indicated a reduction in the extent of pyrimidine dimer and 6-4 photoproduct removal in the DnaK-deficient cells. These results suggest that molecular chaperonins participate in nucleotide excision repair by maintaining repair proteins in their properly folded state.
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INTRODUCTION |
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The UvrA protein is an essential subunit of UvrABC endonuclease (1) and plays an important role in DNA damage recognition and nuclease assembly (2). Binding of UvrA to damaged DNA is specific for a local conformational change at the site of the damaged base (2-4). The primary mechanistic function of UvrA in the UvrABC system is to interact with UvrB to form a heterotrimer (UvrA2B), which helps to locate the site of DNA damage. After dissociation from the UvrA2B-DNA complex, UvrA is believed to load more UvrB proteins onto the site of DNA damage (5).
High concentrations of UvrA have been found to be inhibitory to the incision reaction in vitro (6-8) and can confer UV sensitivity in vivo (9). This phenomenon is due either to competition between UvrA2 and the UvrA2B complex for binding to the damaged DNA, a lack of UvrA2 dissociation from the UvrA2B-DNA pre-incision complex, or to the formation of non-productive dead end complexes (which could be caused by partially denatured or improperly folded proteins). Within the cell, the relative concentrations and the interactions between UvrA and UvrB must be well controlled to allow UvrA to participate in multiple rounds of incision. One group of proteins which often help to facilitate proper protein-protein interactions are the heat shock or chaperone proteins.
Heat shock proteins (Hsp)1 (for reviews, see Refs. 10-14) are involved in many biological processes, including DNA replication (15, 16), SOS mutagenesis (17), transcription as associated with RNAP (18), signal transduction (19-22), polypeptide membrane transport (23-28), and complexing with p53 (29). In these processes, heat shock proteins function in vivo as molecular chaperones to help identify and refold denatured or improperly folded proteins, preventing aggregation. The major heat shock protein families include Hsp104, Hsp90, Hsp70, Hsp60/GroEL, and the small Hsps (10, 30). In Escherichia coli, two heat shock protein families, DnaK (Hsp70) and GroEL (Hsp60), have been widely studied. In particular, DnaK is expressed at a high level, about 1% of the total cellular protein at 37 °C (31). Such abundance, and this conservation throughout procaryotic and eucaryotic organisms, implies a generally important role for heat shock proteins functioning as molecular chaperonins in many biological processes within the cell.
Here we report interactions of UvrA with heat shock proteins of the DnaK, DnaJ, and GrpE family. Our results indicate the following: 1) UvrA is heat-labile; 2) UvrA is stabilized by DnaK or the combination of DnaK, DnaJ, and GrpE under normal in vitro conditions; and 3) UvrA in the presence of DnaK, DnaJ, and GrpE undergoes multiple cycles of UvrB loading, leading to increased incision efficiency; 4) denatured UvrA can be refolded and reactivated by DnaK, DnaJ, and GrpE; 5) a DnaK mutant strain shows decreased survival after UV and a lower extent of photoproduct removal.
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EXPERIMENTAL PROCEDURES |
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UvrA, UvrB, and UvrC Purification-- UvrA was purified from E. coli strain MH1 UvrA containing the overproducing plasmid, pSST10 (graciously supplied by L. Grossman, Johns Hopkins University), in which the uvrA gene is under the control of the heat-inducible PL promoter. UvrB and UvrC were overproduced from E. coli strain CH296 containing plasmids pUNC211 and pDR3274, respectively (graciously supplied by A. Sancar, University of North Carolina). All three proteins were purified to homogeneity as described previously (32).
Heat Shock Proteins-- All heat shock proteins used in this study were purchased from StressGen Biotechnologies.
BPDE-damaged DNA-- The 50-base pair oligonucleotides containing a single BPDE adduct, (+)-cis-anti-BPDE, were prepared as described previously (2). The phosphorylated BPDE-11-mers (30 pmol) (generously supplied by Dr. Nick Geacintov, Department of Chemistry, New York University) were ligated with equal moles of 20-mer (5'-terminally labeled with 32P) and phosphorylated 19-mer in the presence of the complementary strand 50-mer and T4 DNA ligase in a 30-µl solution containing 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, and 50 µg/ml BSA. The ligation was carried out at 16 °C for 12 h. After ligation, products were purified and then reannealed with the non-damaged 50-mer complementary strand and then purified on an 8% polyacrylamide native gel. The double-stranded character and homogeneity of the 50-base pair substrates were examined by a restriction assay (2) and analyzed on a 12% polyacrylamide sequencing gel under denaturing conditions.
Thermal Stabilization Assay-- UvrA (0.25 or 1.0 nM) was incubated in the presence or absence of DnaK or the combination of DnaK, DnaJ, and GrpE at 37 °C for discrete periods of time or at various concentrations, as indicated in the figure legends, in 10 µl of UvrABC buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 5 mM DTT, and 1 mM ATP). After heat inactivation, the 5'-terminally labeled BPDE-DNA substrate (2 nM) with UvrB (200 nM), UvrC (50 nM), and more ATP (1 mM) were mixed into the reaction to initialize the UvrABC-mediated incision of the adduct at 37 °C for 60 min. The reaction was stopped by addition of EDTA to 25 mM, followed by heating at 90 °C for 3 min. The incision products were then analyzed in a 10% (w/v) polyacrylamide sequencing gel under denaturing conditions with TBE buffer (50 mM Tris, 50 mM boric acid, and 1 mM EDTA, pH 8).
Stabilization during Incision-- UvrA (0.25 or 1.0 nM), UvrB (200 nM), and UvrC (50 nM) were first mixed in the absence or presence of heat shock proteins (DnaK, DnaJ, and GrpE 0.9/0.01/0.2 µM) in 60 µl of UvrABC buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 5 mM DTT, and 1 mM ATP). Then the 5'-terminally labeled BPDE-DNA substrate was added immediately to initiate the incision at 37 °C. After the indicated periods of incision, a 10-µl aliquot was withdrawn and heated at 90 °C for 3 min to stop the reaction. The incised products were analyzed in a 10% (w/v) polyacrylamide sequencing gel under denaturing conditions.
UV Survival--
The E. coli strains used in this
study are as follows: MC4100, wild-type cells; BB1553, a
dnaK52 sidB1 mutant; GW8306, BB1553 cells containing
pJM41 plasmid encoding wild-type DnaK
(pBR322-PlacdnaK+); and
GW8304, BB1553 cells harboring the pBR322 plasmid (all strains were
generously provided by G. C. Walker, Massachusetts Institute of
Technology) (33, 51). The sidB1 mutation allows stable
propagation of cells in which dnak has been deleted.
SidB1 is a mutation in rpoH, the gene for
32.
The BB1553 cells have a similar growth rate to that of wild-type cells,
MC4100 (33). MC4100 and BB1553 cells were grown in LB. GW8304 and
GW8306 cells were grown in LB containing 100 µg/ml ampicillin, 60 µg/ml kanamycin, and 0.5 mM IPTG. Cells were grown to
saturation overnight at 30 °C and then diluted by about 200-fold
into the same medium, followed by continuous growth at the same
temperature to an optical density of 0.4 at 600 nm. Cells were
pelleted, washed, and resuspended to the original density in 0.9% NaCl
solution. Irradiation of the cell suspensions was performed with a
germicidal lamp (254 nm) at 100 microwatts for various periods to
damage the cells. The irradiated cells were then appropriately diluted
with 0.9% NaCl solution on ice, plated on LB agar (for MC4100 and
BB1553 cells) or LB agar containing 100 µg/ml ampicillin, 60 µg/ml
kanamycin, and 0.5 mM IPTG (for GW8304 and GW8306 cells),
and incubated at 30 °C in the dark. Plates containing 100-800
colonies were counted to calculate the survival.
Cell Growth and DNA Preparation for DNA Repair Assay-- Cells were grown at 30 °C in Davis medium supplemented with 0.4% glucose, 0.2% casamino acids, and 1 µg/ml vitamin B1. Cultures were grown to mid log phase (approximately 3 × 108 cells/ml) as measured by A600. Cells were collected by filtration on 0.45-mm Millipore filters, washed with prewarmed Davis medium, and resuspended in Davis medium. Cells were UV-irradiated in a glass Petri dish on a rotating platform under a germicidal lamp with a dose of 40 J/m2 ultraviolet-light (254 nm) and placed in a flask containing growth supplements. Samples of the culture were removed at various times and mixed with an equal volume of ice-cold NET (100 mM NaCl, 10 mM Tris, pH 8.0, 10 mM EDTA) buffer. Cells were pelleted by centrifugation at 4 °C, resuspended in TE, pH 8.0, and were lysed by addition of 1 mg/ml lysozyme and 100 mg/ml RNase A and incubation for 15 min at 37 °C. Proteinase K (100 mg/ml) and Sarkosyl (0.5%) were then added, and the mixture was incubated at 50 °C for 1 h. The DNA was extracted with phenol/chloroform and precipitated with 2.5 M ammonium acetate and 2 volumes of 95% ethanol. Purified DNA was resuspended in TE, pH 8.0. The DNA was lightly sonicated using a Branson sonifier, and the concentration was determined by fluorometry using Hoechst 33258.
Immunoassay for Global Nucleotide Excision Repair-- The repair of cyclobutane dimers (CPD) and 6-4 photoproducts (6-4 PP) in each sonicated DNA sample was determined using an immunoassay. Following denaturation by boiling, 25 ng (CPD) or 250 ng (6-4PP) of each DNA sample was loaded in triplicate onto a Hybond N+ membrane using a slot blot apparatus. The membrane was incubated for 2 h in the presence of mouse antibodies against either CPDs (TDM-2) or 6-4 PPs (64M-2) diluted 1:2000 in phosphate-buffered saline (antibodies were a generous gift of Dr. Toshio Mori). Horseradish peroxidase-conjugated secondary antibodies were used at a dilution of 1:5000 and detected using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) and subsequent analysis on phosphor screens (Bio-Rad).
Reactivation Assay-- UvrA protein (126 nM) was inactivated by incubation in 21 µl of 6 M guanidine HCl at 30 °C for 30 min. The denatured protein (0.5 µl) was then diluted 125-fold into the refolding buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 5 mM DTT, 1 mM ATP), with or without the heat shock proteins DnaK, DnaJ, and GrpE (1.0/0.03/0.64 µM) and subsequently incubated at 37 °C for the periods indicated. After the incubation, aliquots of the refolding mixture were added with UvrB (200 nM), UvrC (50 nM), more ATP (1 mM), and the 5'-terminally labeled BPDE-DNA substrate (2 nM) to start the incision reactions. The incision was carried out at 37 °C for 60 min. The incised products were then analyzed in the polyacrylamide sequencing gel under denaturing conditions.
Immunoprecipitation-- UvrA (0.25-5 nM) was first incubated with 145 nM DnaK (StressGen Biotechnologies) at 37 °C for 30 min in 20 µl of IM buffer (50 mM Tris-HCl, pH7.5, 50 mM KCl, 10 mM MgCl2, and 5 mM DTT). Murine monoclonal anti-DnaK (StressGen Biotechnologies) antibody (1 µg) was added to the mixture, which was then incubated with gentle shaking at 4 °C for 1 h. After the incubation, the sample was mixed with 12 µg of swollen protein A-Sepharose 4B fast flow beads (Sigma) and continuously incubated with gentle shaking at 4 °C for another hour. The sample was centrifuged at 12,000 × g at 4 °C for 5 min, and the supernatants were removed. The Immunobeads were washed with IM buffer at 4 °C three times. The beads were mixed with 20 µl of SDS-polyacrylamide gel electrophoresis sample buffer and heated at 90 °C for 5 min. The mixture was loaded onto an 8% (w/v) SDS-polyacrylamide gel electrophoresed for separation. The gel was blotted to a Hybond nitrocellulose membrane (Amersham Pharmacia Biotech) using a Hoefer electrotransfer unit in a buffer containing 10 mM CAPS, pH 11, and 10% MeOH. The membrane was then treated with rabbit anti-UvrA polyclonal following the ECL Western blotting procedures (Amersham Pharmacia Biotech) to identify the UvrA protein bands.
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RESULTS |
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Stabilization of UvrA by Hsp-- Models of the action mechanism of the UvrABC system indicate that UvrA is capable of loading multiple UvrB molecules at damaged sites (1). Whereas UvrA has been shown to turn over on randomly damaged plasmids (5), we have been unable to demonstrate its turnover on defined DNA substrates containing a site-specific BPDE-N2-guanine adduct (2). Since UvrB loading takes several minutes, we reasoned that UvrA at dilute concentrations might be heat-labile, and molecular chaperone proteins might help stabilize UvrA. Fig. 1 shows the thermal stability of UvrA at 37 °C in the absence and presence of all three heat shock proteins (DnaK, DnaJ, and GrpE). The thermal stability of UvrA was assessed by the incision activity of UvrABC system on a DNA substrate containing a single site-specific BPDE adduct. To characterize UvrA specifically under conditions where other components were in excess and would not significantly affect the results, a limited concentration of UvrA was used in our experiments, relative to the DNA substrate and the UvrB and UvrC subunits. As shown in Fig. 1, in the absence of the heat shock proteins, UvrA quickly lost its activity at 37 °C, while the presence of the heat shock proteins greatly protected UvrA from inactivation. Since the lost enzymatic activity was not restored after subsequent incubation with DnaK, DnaJ, and GrpE (data not shown), it appears that the heat leads to the irreversible aggregation of UvrA.
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Reactivation of UvrA by Hsps-- One of the most important characteristics of heat shock proteins is their ability to refold denatured protein substrates into their native structures and to allow restoration of their enzymatic activities. To explore the relationship of UvrA with E. coli heat shock proteins of the Hsp70 family, we conducted UvrA reactivation experiments. To avoid aggregation among the unfolded polypeptides, 6 M guanidine hydrochloride was used instead of thermal denaturation. After the inactivation of UvrA, the denatured protein was diluted 1:125 into refolding buffer containing DnaK, DnaJ, and GrpE to allow restoration of the activity. To determine the background activity, the denatured UvrA was also diluted into the same buffer without heat shock proteins. As shown in Fig. 4, the heat shock proteins were able to restore about 50% of the native incision activity of UvrA within 10 min. As a control, BSA or DnaK, DnaJ, and GrpE in the absence of ATP showed no or little power to reactivate UvrA (Fig. 4b). Individually, the member of the Hsp70 family showed some ability to restore UvrA activity, but the combination of all three provided the highest level of reactivation of unfolded UvrA.
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Immunoprecipitation of UvrA-DnaK Complex--
The reactivation of
UvrA by DnaK, DnaJ, and GrpE strongly suggests a direct interaction of
the heat shock proteins with UvrA. We therefore investigated the
potential binding between denatured UvrA and DnaK in an
immunoprecipitation assay that has been widely used for study of
protein-protein interactions (43). In this assay (see "Experimental
Procedures"), native UvrA was heated at 37 °C in the presence of
DnaK in a buffer containing no ATP. Then DnaK antibody was added and
allowed to bind to DnaK as well as to any potential UvrA-DnaK complex.
Treatment with Immunobeads, which recognize the antibody, followed by
centrifugation, allows the separation of the bead-bound proteins from
the proteins that are free in solution. The immunoprecipitated samples
were analyzed by SDS-polyacrylamide gel electrophoresis and probed with
UvrA antibody (UvrA) on Western blots. As shown in Fig.
5, the identified bands indicate that
DnaK acting as molecular chaperone does bind to UvrA in this assay.
This binding may act to prevent UvrA from denaturing and aggregating
(Fig. 5).
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UV Sensitivity of a DnaK-deficient Strain--
The results from
the in vitro studies suggested a potential role for heat
shock proteins in nucleotide excision repair in vivo. To
help confirm this idea, we conducted in vivo experiments to
study the UV sensitivity of DnaK-deficient cells as compared with
wild-type cells. Fig. 6 demonstrates the
survival of wild-type cells MC4100 and BB1553 mutant (dnaK52
sidB1) (33) versus UV dose. In comparison to wild-type
cells, BB1553 cells displayed significantly more sensitivity to UV
irradiation at various doses and were about 80-fold more sensitive at
100 J/m2. As a control, BB1553 mutant cells carrying the
plasmid, pJM41, which encodes DnaK under an IPTG-inducible
lac promoter showed similar UV resistance as wild-type cells
(Fig. 6). These results demonstrate that the dnaK deletion,
but the sidB1 mutation, conferred UV sensitivity. Since both
wild-type and mutant cells grew at the similar rate at 30 °C (33),
one of the possible explanations for these results is that the DnaK
deletion mutant cells may have a limited capacity for nucleotide
excision repair, due to the reduction of UvrA activity. This hypothesis
was tested below.
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Repair Kinetics of UV Photoproducts in DnaK-deficient Cells-- The increased UV sensitivity exhibited by BB1553 cells could result from a number of different factors including lack of DNA replication resumption or slower kinetics of DNA repair. In order to investigate the latter, BB1553 and MC4100 were irradiated with 40 J/m2 of ultraviolet light (254 nm), and the frequency of pyrimidine dimers and 6-4 photoproducts was determined by DNA slot blot analysis using specific monoclonal antibodies to each photoproduct. The results collected in Fig. 7 indicate that BB1553 cells, DnaK-deficient, had slower rates of repair of 6-4 photoproducts and lower extents of repair for both types of DNA photoproducts. These results are entirely consistent with the in vitro results indicating DnaK is necessary for the turnover of UvrA and that without the action of DnaK in vivo there is a limited number of active UvrA molecules that would lead to lower extents of repair.
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DISCUSSION |
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UvrA is a critical protein in E. coli nucleotide excision repair because of its direct role in damage recognition and in guiding UvrB to the damaged site. In this report we sought to understand why UvrA could not act catalytically during incision reactions on defined substrates containing a site-specific lesion. In the course of these studies we found the following. 1) Heat shock proteins allowed UvrA to participate in multiple rounds of UvrB loading and to act catalytically in the incision reaction. 2) DnaK or the combination of DnaK, DnaJ, and GrpE helped to protect UvrA from thermal denaturation and irreversible inactivation. 3) DnaK interacted directly with denatured UvrA forming a protein-protein complex. 4) Denatured but not aggregated UvrA could be reactivated by DnaK, DnaJ, and GrpE to up to ~50% of the native protein activity. 5) UV survival and repair kinetics in a DnaK-deficient cells indicate an important in vivo role for DnaK in nucleotide excision repair, presumably by its interaction with UvrA.
The dnaK, dnaJ, and grpE genes of
E. coli were first identified by mapping mutations that were
unable to support bacteriophage replication (44). Subsequent
analysis indicated that these genes encode heat shock proteins that are
ubiquitous throughout nature (11, 12). These proteins function as
molecular chaperones to maintain proteins in the properly folded state.
For example, Walker and co-workers (45) have shown that GroE heat shock
protein is absolutely required for SOS-induced mutagenesis, presumably through its stabilization of UmuC.
Whereas earlier studies indicated that UV light and naladixic acid were able to induce DnaK and GroEL in an SOS-independent manner (46), no enhanced survival functions have been attributed to the induction of these proteins. Experiments presented in Fig. 6 clearly show that a DnaK-deletion strain, BB1553, is significantly more UV-sensitive than an isogenic wild-type strain. Furthermore repair kinetics of 6-4 PP and CPD in these two strains (Fig. 7) indicate that loss of DnaK activity leads to a decrease in the overall extent of repair for both CPD and 6-4 PP and a slower rate of repair for 6-4 PP.
These in vivo findings are entirely consistent with our in vitro results that indicate that the addition of DnaK, DnaJ, and GrpE to UvrA leads to increased rate and extent of incision of a defined substrate containing a single BPDE-N2 guanine adduct (Fig. 3). UvrA is extremely heat-labile when in dilute concentrations, with a t1/2 of less than 5 min at 37 °C (Figs. 1 and 2). However, the addition of DnaK either singularly or with DnaJ and GrpE greatly stabilized UvrA to heat denaturation and led to multiple loadings of UvrB.
The data presented in this work demonstrate that DnaK, DnaJ, and GrpE directly interact as molecular chaperones with UvrA to keep UvrA in its native state. It is believed that exposed hydrophobic side chains of denatured proteins are the primary determinants for DnaK binding (47, 48). The analysis of crystal structure of DnaK complexed with a peptide at its substrate-binding site has indicated that the binding of DnaK was centered to its substrate on hydrophobic residues that become completely buried in a deep pocket on DnaK (42). This suggests that DnaK binds to unfolded proteins that expose hydrophobic residues on their surface. In our experiments, UvrA was denatured by heating at 37 °C, and UvrA may have aggregated in the absence of chaperones, resulting in irreversible loss of UvrA enzymatic activity. Incubation of the UvrA aggregate with DnaK, DnaJ, and GrpE failed to recover any UvrA activity (data not shown). In the presence of DnaK, non-native UvrA was protected from aggregation or misfolding most likely by the binding of DnaK to the UvrA.
UvrA after denaturation by guanidine hydrochloride was reactivated up to about 50% of the original activity by DnaK or the combination of DnaK, DnaJ, and GrpE in the presence of ATP. The difference between the heat and guanidine hydrochloride treatments is that the former results in aggregation of UvrA and the latter leads to the formation of unfolded UvrA without aggregation. Our results indicated that although there were no significant differences between DnaK or the combination of DnaK, DnaJ, and GrpE in heat stabilization of UvrA, the combination of all three proteins was evidently more efficient in reactivating unfolded UvrA (Fig. 4). A similar observation was reported for the interactions of DnaK or DnaK, DnaJ, and GrpE with RNA polymerase by Skowyra et al. (18) and Ziemienowicz et al. (49). The difference in reactivation may be attributed to a higher efficiency for DnaK working together with coenzymes of DnaJ and GrpE. DnaJ has been reported to stimulate the ATP hydrolysis of DnaK to form ADP-DnaK intermediate that binds peptide substrate tightly (39, 50). Then GrpE dissociates the ADP from DnaK, resulting in the release of substrate (36, 38).
In conclusion, data presented here clearly show that DnaK stabilizes UvrA from heat inactivation and allows UvrA to act catalytically during in vitro incision reactions. Measurements of in vivo repair kinetics and UV survival of a DnaK-deficient E. coli strain clearly indicate a role for DnaK in nucleotide excision repair. Future studies will be directed toward understanding whether DnaK and other E. coli heat shock proteins can help promote the release of UvrA from the UvrAB-DNA complex and whether these chaperone proteins act on other components of the nucleotide excision repair system. It will be of interest to determine whether heat shock proteins help aid in the proper assembly/disassembly, and thus efficiency, of multi-subunit protein machines involved in eucaryotic nucleotide excision repair.
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ACKNOWLEDGEMENTS |
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We appreciate the critical reading of this manuscript by Drs. Randy Walker, David Konkel, and Heather Bassett. We also thank Dr. Nick Geacintov (New York University) for the generous support and encouragement. We acknowledge Brenda Romero for helping prepare this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant ES 7955 (to B. V. H.) and NIEHS Center Grant ES06676 (to R. S. Lloyd).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be sent: Sealy Center for Molecular Science, 5.138 MRB 1071, University of Texas Medical Branch, Galveston, TX 77555-1071. Tel.: 409-772-2144; Fax: 409-772-1790; E-mail: bvanhout{at}SCMS.utmb.edu.
1 The abbreviations used are: Hsp, heat shock protein(s); BPDE, benzo[a]pyrene-diol epoxide, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BSA, bovine serum albumin; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; CPD, cyclobutane pyrimidine dimer; DTT, dithiothreitol; IPTG, isopropylthiogalactopyranoside; 6-4 PP, 6-4 photoproduct.
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REFERENCES |
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