Two Forms of UvrC Protein with Different Double-stranded DNA Binding Affinities*

Moon-shong TangDagger, Michael Nazimiec, Xiangcang Ye, Ganesh H. Iyer, Jamie Eveleigh, Yi Zheng, Wenjing Zhou, and Yen-Yee Tang

From the Department of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987

Received for publication, September 18, 2000, and in revised form, October 27, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using phosphocellulose followed by single-stranded DNA-cellulose chromatography for purification of UvrC proteins from overproducing cells, we found that UvrC elutes at two peaks: 0.4 M KCl (UvrCI) and 0.6 M KCl (UvrCII). Both forms of UvrC have a major peptide band (>95%) of the same molecular weight and identical N-terminal amino acid sequences, which are consistent with the initiation codon being at the unusual GTG site. Both forms of UvrC are active in incising UV-irradiated, supercoiled phi X-174 replicative form I DNA in the presence of UvrA and UvrB proteins; however, the specific activity of UvrCII is one-fourth that of UvrCI. The molecular weight of UvrCII is four times that of UvrCI on the basis of results of size exclusion chromatography and glutaraldehyde cross-linking reactions, indicating that UvrCII is a tetramer of UvrCI. Functionally, these two forms of UvrC proteins can be distinguished under reaction conditions in which the protein/nucleotide molar ratio is >0.06 by using UV-irradiated, 32P-labeled DNA fragments as substrates; under these conditions UvrCII is inactive in incision, but UvrCI remains active. The activity of UvrCII in incising UV-irradiated, 32P- labeled DNA fragments can be restored by adding unirradiated competitive DNA, and the increased level of incision corresponds to a decreased level of UvrCII binding to the substrate DNA. The sites of incision at the 5' and 3' sides of a UV-induced pyrimidine dimer are the same for UvrCI and UvrCII. Nitrocellulose filter binding and gel retardation assays show that UvrCII binds to both UV-irradiated and unirradiated double-stranded DNA with the same affinity (Ka, 9 × 108/M) and in a concentration-dependent manner, whereas UvrCI does not. These two forms of UvrC were also produced by the endogenous uvrC operon. We propose that UvrCII-DNA binding may interfere with Uvr(A)2B-DNA damage complex formation. However, because of its low copy number and low binding affinity to DNA, UvrCII may not interfere with Uvr(A)2B-DNA damage complex formation in vivo, but instead through double-stranded DNA binding UvrCII may become concentrated at genomic areas and therefore may facilitate nucleotide excision repair.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The initial events of nucleotide excision repair in Escherichia coli are controlled by three uvr gene products: UvrA, UvrB, and UvrC proteins. Our current understanding suggests that these three proteins work in a sequential manner, with UvrA first locating and dimerizing at a damaged base on double-stranded DNA (ds-DNA),1 and UvrB then binds and forms a Uvr(A)2B complex from which UvrA may dissociate. Finally, UvrC binds to the Uvr(A)2B complex and triggers dual incisions 6-8 base pairs (bp) 5' and 3-5 bp 3' to the damaged base. A diverse array of chemically and photochemically induced modifications to bases in DNA are recognized, including helix-stabilizing and -destabilizing damage (for review, see Refs. 1-3).

Recent studies show that UvrC is essential for 5' and 3' incisions and is specifically implicated in making the 5' incision through site-directed mutagenesis studies. UvrC is probably essential for the catalysis of the 3' incision as well, although it may catalyze this step itself (4-11).

uvrC differs from uvrA and uvrB in its genomic organization. It may not be under lexA control as uvrA and uvrB are (7). The genomic region of E. coli containing the uvrC gene is complex and contains at least one overlapping open reading frame (12, 13). The uvrC gene itself appears to have four promoters and to be transcribed into two mRNAs (7). The translation start site for the UvrC protein is also unclear and may be the 5'-GTG upstream of the first 5'-ATG (13, 14). The precise size of the UvrC protein is therefore unknown and may be 67,000 Da, or it may be 22 amino acids longer and 68,500 Da.

The low natural occurrence of the Uvr proteins has necessitated their study by the use of overproducing cells containing the appropriate plasmids. This is particularly true for UvrC, because it is estimated to be present at only <10 molecules per wild-type cell (2).

In this study we have purified UvrC from E. coli strains containing the overproducing plasmid pDR3274 (15) by several different methods, including a rapid procedure that avoids precipitation of the protein and expedites isolation of a purified protein. We find that these preparations yield two different UvrC fractions, which we call UvrCI and UvrCII according to their elution positions from single-stranded DNA (ss-DNA) cellulose columns. Both UvrCI and UvrCII are active in a variety of assays of UvrABC excision nuclease activity but were found to differ in their interactions with DNA. UvrCII binds readily to ds-DNA, whereas UvrCI does not. We present evidence that demonstrates that this UvrCII-DNA binding may affect the UvrABC excision nuclease activity. These two forms of UvrC were also isolated from wild-type E. coli cells without containing the pDR3274 plasmids (12). The possible molecular differences between these two forms of the UvrC protein and their physiological roles are discussed.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Restriction enzymes HinfI, NarI, EoRI, and PstI, T4 polynucleotide kinase, bacterial alkaline phosphatase, acrylamide, bisacrylamide, agarose, and NACS Prepacs convertible columns (NACS PACS) were obtained from Life Technologies, Inc. The restriction enzyme BstNI was obtained from New England Biolabs. Yeast tRNA was obtained from Sigma. All 32P-labeled nucleotides were obtained from Amersham Pharmacia Biotech NEN Life Science Products, Inc. Nitrocellulose membranes (hydrophilic, 0.45 µM, HAWP) were purchased from Millipore.

DNA and DNA Fragment Isolation-- Phi X174 replicative form I (RFI) DNA and plasmid pBR322 were isolated and purified by cesium chloride density gradient centrifugation. T7 phages were prepared by a method described by Yamamoto and Alberts (16). T7 DNAs were prepared by removing proteins from phages by phenol and diethyl ether extractions. The 247-bp HinfI-BstNI single 3' end-32P-labeled fragments of pBR322 were isolated from a 383-bp BstNI fragment that had been agarose gel-purified and 3' end-labeled with [alpha -32P]dTTP. The 174-bp EcoRI-HaeIII single 5' end-32P-labeled fragments of pBR322 were prepared as described previously (17). The EcoRI-PstI 750-bp fragments of pBR322 were isolated from agarose gel electrophoresis and 32P-labeled with [gamma -32P]ATP at both 5' ends.

UV Irradiation-- Phi X174 RFI DNA, [3H]thymidine-labeled T7 DNA, and 32P-labeled defined DNA fragments were irradiated with a germicidal lamp (Sylvania, C15T8; major emission, 254 nm) to produce eight dimers or one dimer per DNA molecule.

Purification of Uvr Proteins-- UvrA, UvrB, and UvrC proteins were purified from the E. coli K12 strain CH296 (uvrC34)- and DR1984 (recA1 uvrC34)-carrying plasmids pUNC45 (uvrA), pUNC211 (uvrB), or pDR3274 (uvrC; Ref. 16). These plasmids and E. coli strains were kindly provided by Dr. A. Sancar (University of North Carolina, Chapel Hill, NC). The pUVC1234 plasmid construct (12), which contains the endogenous uvrC operon, was a generous gift from Dr. R. Moses (University of Oregon, Portland, OR). UvrC protein was also purified from E. coli cells (MST 1) with the wild-type uvrC gene (17, 18).

UvrABC Excision Nuclease Reactions-- The UvrABC excision nuclease reaction was conducted in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM KCl, 1 mM ATP, and 1 mM dithtiothreitol. An aliquot of 32P-labeled DNA or phi X174 RFI DNA (0.2 µg) was reacted with 15 nM UvrA, 15 nM UvrB, and 15 nM UvrC in a volume of 25 µl for 60 min at 37 °C. For phi X174 RFI DNA the reactions were stopped by adding 0.1% SDS and heating at 65 °C for 5 min, and the DNAs were then electrophoresed in a 1% agarose gel in buffer containing 40 mM Tris acetate, pH 8.0, and 1 mM EDTA at 1 V/cm for 14 h. The gel was stained with 0.5 µg/ml ethidium bromide to visualize the supercoiled and relaxed forms of DNA. The pictures were scanned by a Bio-Image Analyzer with a Visage 100 whole-band analysis software program. For 32P-labeled DNA fragments the reactions were terminated by phenol extractions. The labeled DNA was then ethanol precipitated, washed in 75% ethanol, dried, and dissolved in a formamide denaturing dye mix (80% v/v formamide, 0.1% xylene cyanol, and 0.1% bromphenol blue).

Sequencing Gel Electrophoresis-- Chemical sequencing was carried out as described by Maxam and Gilbert (21) with the modifications described (17). DNA samples were heated at 90 °C (2 min) and quenched in an ice bath. The samples were applied to a sequencing gel, consisting of 8% acrylamide and 7 M urea in Tris borate-EDTA buffer (50 mM Tris-HCl, 50 mM sodium borate, and 10 mM EDTA, pH 8.3), in parallel with the Maxam and Gilbert (21) sequencing reactions. After electrophoresis the gel was dried in a Bio-Rad gel dryer and exposed to Eastman Kodak Co. X-Omat RP films at -70 °C for various lengths of time. The intensity of bands was determined by scanning with a Bio-Image Analyzer as described above.

DNA Binding Assay-- 32P-labeled pBR322 DNA fragments or linearized phi X174 RFI DNA, and [3H]thymidine-labeled T7 DNA were incubated with UvrC or UvrA at different protein/DNA ratios in UvrABC excision nuclease reaction buffer for 60 min at 37 °C. At the end of incubation the mixtures were chased with an excessive amount of calf thymus DNA for 10 s, filtered through a membrane (0.45 µM; HAWP, Millirpore), and washed with UvrABC excision nuclease reaction buffer without ATP for 10 s or electrophoresed in a 0.5% agarose gel in buffer containing 50 mM Tris, pH 7.9, 50 mM acetate, and 1 mM EDTA. The radioactivity in the dried membranes was counted in an LKB 1219 scintillation counter. The agarose gels were air-dried and exposed to Kodak X-Omat RP films at -70 °C for various times. The intensity of bands was determined by scanning with a Bio-Image Analyzer.

N-terminal Amino Acid Analysis-- To determine the N-terminal amino acid sequence of UvrC, the proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane. The UvrC band was eluted, and the first 12 N-terminal amino acids were determined by Beckman amino acid sequences according the method as described by Matsudaira (22).

UvrC Cross-link Reactions-- Aliquots containing 1 µg of UvrC were incubated with 0.1% glutaraldehyde at room temperature for 10 min. Immediately after the reaction the proteins were denatured by boiling in SDS solution for 5 min, separated by 7% SDS-PAGE, and then transferred to a nitrocellulose membrane.

Immunoblotting-- 7 or 10% acrylamide, 0.32% bisacrylamide, and SDS were used as described by Laemmli (23). After electrophoresis the proteins were electrotransferred to a nitrocellulose membrane and reacted with polyclonal UvrC antiserum (18); the antigen-antibody complex was further conjugated with horseradish peroxidase-labeled antibodies and then detected by chemiluminescence (24).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of Two Forms of UvrC by ss-DNA Cellulose Chromatography-- Several purification schemes have been reported for UvrC purification, and they have in common two steps: phosphocellulose and ss-DNA-cellulose chromatography (15, 25, 26). UvrC proteins can be precipitated with polymin P before going through chromatographic separation (25). In these procedures UvrC is eluted last from an ss-DNA-cellulose column with 0.65 M KCl. We found that substituting a KCl gradient for the more usual step gradient to elute the ss-DNA-cellulose column results in the elution of two distinct peaks of A280 material, and both peaks have UvrC activity (Fig. 1A; details are described in the following sections).



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Fig. 1.   Separation of two forms of UvrC by ss-DNA-cellulose chromatography. The UvrC proteins from cell lysates of CH296/pDR3274 were either precipitated with polymin P (0.5%; A) or left without this precipitation (B). Partially purified UvrC collected from combined peak fractions of phosphocellulose chromatography was diluted to 0.2 M KCl in buffer A and applied to a 1.5 × 14-cm column containing 0.5-1 g ss-DNA-cellulose. The column was washed and developed with a linear gradient of KCl (0.3-1 M KCl; dotted lines) at a flow rate of 0.5 ml/min, and the 4-ml fractions were collected. A and B, wavelength 280-nm absorbance profile of the eluted fractions (solid lines). C, SDS-PAGE analysis of proteins in the different fractions from B. Lane 1, molecular standards; their molecular masses (in kilodaltons) are indicated at the left.

Because the two-peak elution profile of the UvrC protein has never been reported before, we then explored the possibility of whether the multistep purification procedure for UvrC was responsible for this striking pattern by simplifying the purification scheme. UvrC was purified from a cell sonicate (CH296 or DR1984 cells containing pDR3274 and induced with 0.5 mM isopropyl beta -D-thiogalactoside) prepared in 0.3 M KCl. The supernatant after centrifugation was purified through a phosphocellulose column and then an ss-DNA-cellulose column without going through a polymin P precipitation step. Elution of the ss-DNA-cellulose column with a KCl gradient gives a similar profile as in Fig. 1A, showing two peaks of protein (Fig. 1B). Both peaks contain a major (>95%) UvrC band of a molecular mass of 65,000 Da when separated by SDS-PAGE (Fig. 1C); the amount of UvrC collected in the first peak fractions is approximately twice as much as in the second peak fractions. We refer to the two fractions of UvrC according to their elution positions as UvrCI (0.4 M KC1) and UvrCII (0.6 M KC1).

UvrCI and UvrCII Are Active in Incising UV-irradiated, Supercoiled phi X174 RFI DNA-- The activity of these two forms of UvrC in incising UV-induced DNA damage was tested. Various amounts of UvrC proteins were reacted with UV-irradiated phi X174 RFI DNA (2.2 nM) in the presence of UvrA (15 nM) and UvrB (15 nM). (We term this reaction condition, which has a relatively high nucleotide/Uvr ratio, condition I). The results in Fig. 2 show that both forms of UvrC were active in incising UV-irradiated, supercoiled DNA. However, the specific activity of UvrCII is lower than that of UvrCI; the former is one-fourth as active in incising UV-irradiated phi X174 RFI DNA as the latter.



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Fig. 2.   UvrABCI and UvrABCII nuclease incisions of UV-irradiated supercoiled DNA. Supercoiled Phi X174 RFI DNA containing eight pyrimidine dimers per DNA molecule (U8) was reacted with various amounts of either UvrCI or UvrCII (1 represents 15 nM; the molar concentration was calculated on the basis of the assumption that the molecular mass of both UvrC forms is 65 kDa according to the results shown in Fig. 1C) in combination with UvrA (15 nM) and UvrB (15 nM) under standard reaction conditions at 37 °C for 60 min, and the resultant DNA was separated by electrophoresis in a 1% agarose gel. A, typical gel. Top panel, individual Uvr proteins used in the incision assay. U0, unirradiated Phi X174 RFI DNA; +, Uvr protein presence; -, Uvr protein absence; CCC, covalently closed circle; OC, open circle. B, effect of UvrC concentration on the incision of U8 DNA. The fraction of U8 incised was quantified from densitometer scanning of bands corresponding to CCC and OC in A.

Because the majority of proteins present in both peaks have a molecular mass (denatured by boiling in SDS solution) corresponding to that of UvrC, their N-terminal amino acid sequences were determined. We have found that the first 12 of the 13 N-terminal amino acids of both forms of UvrC are the same; starting from the second amino acid the sequence is: Asp-Gln-Phe-Asp-Ala-Lys-Ala-Phe-Leu-Lys-Thr-Val.

UvrCII Is a Tetramer, and UvrCI Is a Monomer-- Because the monopeptides of UvrCI and UvrCII not only have the same molecular mass but also have the same N-terminal amino acid sequence, and no other peptides have been found in association with these two forms of proteins (Fig. 1), the results raise the possibility that these two forms of UvrC could result from different folding or that the native form of these two proteins may be composed of different numbers of monopeptides, or both. To test these possibilities we have determined the molecular mass of these two forms of UvrC proteins by glutaraldehyde cross-link reaction and size exclusion chromatography (27). Fig. 3 shows that treatment of UvrCI with 0.1% glutaraldehyde resulted in one additional band corresponding to molecular mass of 130 kDa besides the 65-kDa band. In contrast, the same glutaraldehyde treatment of UvrCII resulted in two additional bands; one corresponding to 130 kDs, the same as observed with UvrCI treatment, and the other having a molecular mass >240 kDa. Results from size exclusion chromatography show that UvrCI eluted at a major peak corresponding to the bovine albumin position (68 kDa). In contrast, the UvrCI-UvrCII mixture eluted at two peaks corresponding to ~68 and >232 kDa, respectively (Fig. 3B). Together, these results suggest that UvrCI is a monomer, and UvrCII is a tetramer.



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Fig. 3.   Determinations of UvrCI and UvrCII molecular mass by size exclusion chromatography and glutaraldehyde cross-linking reactions. A, typical gel filtration profile of UvrCI and UvrCII. UvrCI and UvrCII purified from an ss-DNA-cellulose column were separated by Sephacryl S-300 HR gel chromatography (eluted with 0.55 M KCl); a, separation of a mixture of UvrCI and UvrCII; b, UvrCI only; c, UvrCII only. The positions of the molecular mass standards are indicated by arrows. B, typical Western blotting profile of UvrCI and UvrCII after glutaraldehyde treatment. UvrCI and UvrCII proteins were reacted with glutaraldehyde, denatured as described, separated by 7% PAGE, and transferred to a nitrocellulose membrane. The membrane was reacted with UvrC antibodies followed by horseradish peroxidase antibodies. The antigen-antibody reactions were detected by the chemiluminescence method (24). Lane 1, UvrCI mock treated; lane 2, UvrCI treated with 0.1% glutaraldehyde at room temperature for 10 min; lane 3, UvrCII mock treated; lane 4, UvrCII treated with glutaraldehyde as in lane 2. The positions of the molecular standards (in kilodaltons) are indicated.

UvrCI and UvrCII React to UV-irradiated Linear DNA Differently-- It has been well established that the UvrABC excision nuclease makes dual incisions 6-8 bp 5' and 3-5 bp 3' to UV-induced pyrimidine dimers and other chemical-DNA adducts (for review, see Refs. 1-3). However, it has been found that occasionally the 5' and 3' incisions induced by UvrABC may uncouple, and the uncoupled incision occurs in UV-, bulky chemical carcinogen-, and CC-1065-modified DNA (for review, see Ref. 2). Selby and Sancar (28) have reported that "aged" UvrC proteins may lose their 5' incision ability. To determine whether UvrCI and UvrCII, in combination with UvrA and UvrB (we term the collective function of Uvr proteins UvrABCI and UvrABCII excision nuclease, respectively), would have the same dual incision activity, UV-irradiated single 3'-end-32P-labeled HinfI-BstNI 247-bp pBR322 DNA fragments (1 nM) or single 5'-end-32P-labeled EcoRI-HaeIII 174-bp pBR322 DNA fragments were reacted with 15 nM of these proteins (we term this reaction condition, which has a relatively low nucleotide/Uvr ratio, condition II). The results are shown in Fig. 4; although UvrABCI makes the expected dual incisions at pyrimidine dimers, under the same reaction conditions UvrABCII does not incise the same UV-irradiated linear DNA fragments (Fig. 4, A, compare lanes 11 and 15, and B, compare lanes 12 and 17).



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Fig. 4.   A, UvrABCI and UvrABCII incisions of UV-irradiated (one pyrimidine dimer per DNA fragment; lanes 11-18) and unirradiated (lanes 1-6) 3' end-32P-labeled 247-bp BstNI-HinfI pBR322 DNA fragments. The DNA fragments were incubated in the standard reaction mixture containing 15 nM UvrA, 15 nM UvrB, and 15 nM UvrCI or UvrCII and with different amounts of competitive DNA (unirradiated, nonlabeled linear Phi X174 RF ds-DNA as indicated at the top): lanes 11 and 15, 0 µg; lanes 12 and 16, 0.1 µg; lanes 13 and 17, 1 µg; lanes 14 and 18, 2 µg. Lanes 1-6, reactions using unirradiated DNA fragments. The mixtures were incubated at 37 °C for 60 min, and the resultant DNAs were electrophoresed in a sequencing gel. The Maxam and Gilbert (21) A+G, G, T+C, and C sequencing reactions are indicated. Uvr protein treatments were as follows: Lane 1, no enzyme; lane 2, UvrA only; lane 4, UvrCI; lane 3, UvrCII; lanes 5 and 11-14, UvrABCI; lanes 6 and 15-18, UvrABCII. B, UvrABCI and UvrABCII incisions of UV-irradi- ated (one pyrimidine dimer per DNA fragment; lanes 12-21) 5' end-32P-labeled 174-bp EcoRI-HaeIII pBR322 DNA fragments. The conditions for UvrABC reactions and the sequencing gel electrophoresis are the same as described in A. The amounts of competitive DNA added are indicated at the top. The pyrimidine tracts (lanes 1-16), which contain two or more contiguous pyrimidines and which have the potential to form photodimers, are indicated in the left panel. The photodimers induced corresponding UvrABC incision bands (u1-u16) are indicated in the right panel.

There are two possible explanations for these unexpected results; one is that UvrABCII is inactive in incising photodimers in linear DNA, and the other is that UvrCII may interfere with Uvr(A)2B-photodimer complex formation. It has been reported that the presence of excessive UvrA protein inhibits UvrABC excision activity, and the inhibition has been speculated to result from the binding of excessive UvrA proteins to damaged bases, which consequently prevents proper Uvr(A)2B-DNA damage complex formation (29, 30). The inability of UvrABCII to incise the small quantity of UV-irradiated linear DNA fragments shown in Fig. 4 cannot be attributable to an excessive amount of UvrA proteins, because the active UvrABCI reaction conditions contain the same amount of UvrA proteins as in the inactive UvrABCII reaction conditions. One of the major differences between conditions I and II is that although the nucleotide/UvrC ratio is 770 in condition I, it is between 10 and 17 in condition II; the difference between these two conditions is 45-77-fold. If the UvrCII protein is able to bind to ds-DNA and UvrCI is not, then the formation of a proper Uvr(A)2B-DNA damage complex may be affected by excessive amounts of UvrCII (but not UvrCI), similar to the effect of excessive UvrA proteins. To test this possibility we added different amounts of unirradiated, linearized phi X174 RF DNA (competitive DNA) in condition II to reduce the protein/nucleotide ratio. The results in Fig. 4 show that although additional DNA, ranging from 0.05 to 0.2 µg, enhances UvrABCI excision activity, it affects UvrABCII incision activity more drastically, restoring the incision activity of UvrABCII to a level comparable with that of UvrABCI. These results also show that UvrABCII, in the presence of competitive DNA, makes dual incisions 6-8 bp 5' and 3-4 bp 3' of a photodimer in the substrate DNA in the same manner as UvrABCI does. It is worth noting that in the presence of excessive competitive DNA the incision activities of both UvrABCI and UvrABCII are reduced (Fig. 4, A, lanes 14 and 18, and B, lanes 16 and 21).

UvrCII Is a Double-stranded DNA-binding Protein, and UvrCI Is Not-- The above results suggest that UvrCII may be a ds-DNA-binding protein, whereas UvrCI is not. To test this possibility the conventional nitrocellulose filter binding assay for two forms of UvrC and UvrA proteins was performed. Different amounts of Uvr proteins were added to a fixed amount of 3H-labeled T7 DNA (7.3 fmol) with or without UV irradiation to produce 8 dimers per DNA fragment. The protein-DNA mixtures were incubated in the standard UvrABC reaction solution for 60 min at 37 °C and then were chased with an excessive amount of cold DNA before filtering through nitrocellulose membranes. Results in Fig. 5 show that UvrCII indeed binds to ds-DNA, whereas UvrCI does not, and UvrCII-DNA binding is linearly proportional to UvrCII concentrations. Furthermore, it appears that there is no significant difference between the binding affinities of UvrCII to UV-irradiated and unirradiated DNA. These two features are in great contrast to UvrA-DNA binding, which is exponentially proportional to UvrA concentrations and shows higher affinity toward UV-irradiated DNA than nonirradiated DNA (31). Consistent with these filter binding assay results, Fig. 6 shows that the presence of UvrCI proteins does not affect the mobility of the DNA fragments; in contrast, the presence of UvrCII proteins retards the mobility of the labeled DNA fragments significantly. Scatchard plotting (with the assumption that UvrCII is a tetramer with a molecular mass of 272 kDs on the basis of cross-linking reaction results) renders the binding constant of UvrCII-ds-DNA of 9 × 108 M-1 (Fig. 6), which is <FR><NU>1</NU><DE>10</DE></FR>-<FR><NU>1</NU><DE>12</DE></FR> that of UvrA. It is worth noting that a significant portion of the retarded DNA fragments distributes in a smear (in an overly exposed film; data not shown); the smear may result from dissociation of UvrCII from DNA during electrophoresis.



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Fig. 5.   DNA binding of UvrCI (triangle  and black-triangle), UvrCII ( and black-square) and UvrA (open circle  and ). Different concentrations of proteins were incubated with a fixed amount of [3H]thymidine-labeled T7 DNA (7.3 fmol) with (filled symbols) or without (open symbols) UV irradiation (to produce eight dimers per DNA molecule) in the standard UvrABC reaction solution for 60 min at 37 °C. At the end of incubation an excessive amount of calf thymus DNA was added for 10 s, and the mixture was filtered through a 0.45-µm nitrocellulose membrane. DNA retained on the membrane was measured and is interpreted to be protein-bound. All data points are the average of duplicate assays.



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Fig. 6.   A, gel electrophoresis of DNA fragments reacted with UvrCI and UvrCII. Aliquots containing 32P-end-labeled EcoRI-PstI 750-bp pBR322 DNA fragments (14 fmol) were incubated with different amounts of UvrCI (0, 84, 168, 336, and 672 fmol) or UvrCII (0, 84, 168, 252, 336, 420, 504, and 672 fmol) under the same conditions as described in Fig. 5. At the end of the incubation the protein-DNA mixtures were immediately electrophoresed in a 0.5% agarose gel in Tris borate-EDTA buffer. The autoradiographs were scanned, and the binding data were plotted as shown in B. Data points were obtained from results of both the gel retardation assay and the filter binding assay. Inset, Scatchard plot of the binding data (from the gel retardation assay). The average number of UvrCII bound per DNA fragment (upsilon ) was calculated from the distribution of DNA in the various bands as a direct average: upsilon  = Sigma nfn/Sigma fn, where fn is the fraction of DNA fragments that has n proteins bound (31). The percentage of DNA bound in the filter binding assay was normalized by using the highest binding as 100%.

Relationship between UvrCII-DNA Binding and UvrABCII Incision Activity-- To determine the effect of UvrCII-DNA binding on UvrABCII incision activity, 3' end-32P-labeled, UV-irradiated HinFI-BstNI 247-bp DNA fragments (1 nM) were incubated with 15 nM UvrCII in standard reaction conditions for 60 min at 37 °C, different amounts of competitive DNA (nonlabeled linearized phi X174 RF DNA) were added, and the mixtures were further incubated for another 60 min at 37 °C with or without UvrA and UvrB proteins. At the end of the incubation the sites and the extent of incision of these DNAs by UvrABCII (Fig. 7) and the percentage of labeled DNA binding to UvrCII (Fig. 8) were determined by sequencing gel electrophoresis and filter binding assay, respectively. The incision activity of UvrABCII increases as the amount of the additional competitive DNA increases (Fig. 7). Conversely, the results in Fig. 8 show that the fraction of damaged DNA bound with UvrCII decreases exponentially as a function of the concentrations of the competitive DNA. The relationship between the reduction of damaged DNA bound with UvrCII and the increase of UvrABCII incision is better demonstrated in Fig. 8, which shows that the incision reaches a plateau level when UvrCII-damaged DNA binding is reduced to a few percentiles, and further addition of competitive DNA slightly reduces incision. These results are consistent with the interpretation that the UvrCII-DNA binding hinders Uvr(A)2B-DNA damage complex formation and consequently reduces UvrABC incision activity.



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Fig. 7.   Effect of unirradiated, competitive DNA on the incision of UV-irradiated substrate DNA by UvrABCII excision nuclease. The 3'-end-labeled, UV-irradiated 247-bp BstNI-HinfI pBR322 DNA fragments (1 nM) were reacted with UvrCII (15 nM) in the standard UvrABC reaction solution in the presence of different amount of competitive DNA (as indicated at the top) for 60 min at 37 °C. At the end of the incubation the mixtures were either filtered through a nitrocellulose membrane as described in Fig. 5 or further incubated for 60 min after the addition of UvrA (15 nM) and UvrB (15 nM). The resultant DNAs were separated by electrophoresis in a sequencing gel as described in Fig. 4. The symbol and lane descriptions are the same as in Fig. 4.



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Fig. 8.   Relationship between UvrCII-DNA binding and UvrABCII incision of UV-irradiated substrate DNA. The percentage of 32P-labeled, UV-irradiated DNA fragments retained in the filter attributable to UvrCII binding was calculated as described in Fig. 6. The percentage of incision was calculated from the densitometer scanning results of Fig. 7 (total intensity of u1-u16) using the highest cutting as 100% (Fig. 7, lane 17).

Isolation of UvrCI and UvrCII from E. coli Cells with and without Plasmids Containing the Endogenous uvrC Operon-- Because pDR3274 is a recombinant plasmid with the uvrC structural gene sequence linked to the tac promoter (15), it was possible that the two forms of UvrC proteins produced by this plasmid may be different from that produced by the native endogenous uvrC operon. To test this, we isolated UvrC, from E. coli cells without harboring the pDR3274 plasmid. Cell lysates were chromatographed through phosphocellulose and ss-DNA-cellulose columns following the procedures described above. Although the wild-type cell lysates did not render two clear A280 absorbance peaks corresponding to 0.4 and 0.6-0.7 M KCl, Western blotting results show that two peaks of UvrC protein were separated and eluted at these two KCl concentrations (Fig. 9). Together, these results suggest that two forms of UvrC are produced by the endogenous uvrC operon.



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Fig. 9.   Purification of two forms of UvrC from wild-type E. coli cells harboring no uvrC plasmids. A typical immunoblotting result is shown. The purification procedures were the same as described in Fig. 1. The proteins eluted from an ss-DNA-cellulose column by a KCl gradient were separated by electrophoresis using 12% SDS-PAGE, electroblotted to a nitrocellulose membrane, and detected using UvrC antibodies as by described in Fig. 3. UvrC proteins were eluted at fractions at ~0.4 M KCl and at fractions between 0.6 and 0.7 M KCl with no protein detected in the intermediate salt range.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The functions of uvrC are the least understood among the three uvr genes, uvrA, uvrB, and uvrC, which are involved in the initial incision step of nucleotide excision repair. Although in vitro it has been demonstrated that the addition of UvrC proteins to a Uvr(A)2B-DNA damage complex induces dual incisions 5' and 3' to damaged bases, several laboratories have reported that UV irradiation induces DNA single-strand breaks in uvrC34 mutant cells but not in uvrA and uvrB mutant cells, even though these three uvr mutant cells are unable to excise cyclobutane pyrimidine dimers (19, 32-34). Using a viral E. coli transfection system we have found that only uvrC mutant cells show low transfectivity for viral DNA containing N-(guanosin-8-yl)-2- aminofluorene adducts; uvrA and uvrB cells show the same transfectivity as do wild-type cells. In contrast, these three mutant cells have the same low transfectivity for UV-irradiated or N-acetoxy-2-acetylaminofluorene and anti- and syn-benzo(a)pyrene diol epoxide-modified viral DNA (20, 35). These results indicate that uvrC gene products may function in a manner more complicated than simply participating in the incision step and may interact with proteins other than UvrA and UvrB in vivo.

The understanding of UvrC function has been hampered by the scarcity of these proteins in cells and their instability. Purification of UvrC proteins has been achieved by using lambda  lysogens with the uvrC gene (36, 37) or cells with a plasmid containing the uvrC gene (12). The method of purification is mainly based on the ability of UvrC proteins bind to ss-DNA. Yeung et al. (26) have eluted purified UvrC protein at 0.45 M KCl; however, using a step gradient, Thomas et al. (15) have reported that UvrC proteins were eluted completely at 0.65 M KCl. Our modified method further separated the UvrC into two peaks, one at 0.4 M KCl and another at 0.6 M KCl. It appears that the quantity of ss-DNA-cellulose used for column chromatography and the elution rate are the two critical factors for separating these two forms of UvrC proteins; we observed a single UvrC peak, which contained both UvrCI and UvrCII activities when the purification procedure involved a small quantity of ss-DNA-cellulose and a fast elution rate (data not shown).

We have found that these two forms of UvrC proteins can be further purified by ds-DNA-cellulose chromatography but with low recovery (20%); the reason for this low recovery is not clear. However, we also have found that the two forms of UvrC prepared by ss-DNA-cellulose chromatography and those further purified by ds-DNA-cellulose chromatography have the same mobility in SDS-PAGE and the same pI (native form, 7.48; denatured form, 9.4), and in the presence of UvrA and UvrB, both showed a dual incision pattern on UV-irradiated DNA. The sources causing the differences in the ds-DNA binding and elution pattern in ss-DNA chromatography are unknown. Contrary to published results, we have found that the N termini of these two forms of UvrC proteins we have purified are not blocked, and the first 12 of the 13 N-terminal amino acids, starting from the second amino acid, are consistent with the result reported by Moolenaar et al. (13) that the initiation codon for the uvrC gene starts at the 5'-GTG of positions 882-884 rather than at the 5'-ATG of positions 772-774 (14). The source of this discrepancy is unclear.

Because the peptide in the two forms of UvrC have the same molecular mass and N-terminal amino acid sequence, it is possible that UvrCII may have resulted from aggregation of UvrCI. Although we are unable to exclude this possibility entirely, several lines of evidence suggest this may not be the case. First, so far we are unable to convert either form of UvrC to the other by treatments such as oxidation and reduction. Second, we show that only monomeric and tetrameric, but no dimeric and trimeric, UvrC proteins are obtained by size exclusion chromatography. Third, in the electrophoretically separated products of UvrCII treated with the cross-linking reagent glutaraldehyde, we observed mainly the monomeric and tetrameric, but no trimeric, forms of UvrC. Furthermore, we observed the existence of these two forms of UvrC in wild-type cells, even though the copy number of the UvrC proteins in these cells is very low (~10 copies per cell).

It is possible that the difference in ds-DNA binding resides in post-translation modification or in the association of cofactors, or both. If one UvrC form is modified or associated with some cofactors and the other is not, then the two UvrC proteins most likely would be observed in overproducing cells for the simple reason that overproduced UvrC proteins oversaturate the post-translation modification capacity or the amount of cofactors present in the cells. However, using the same purification protocol, we have isolated UvrC forms that eluted at 0.4 M KCl as well as at 0.6-0.7 M KCl from wild-type E. coli cells harboring no recombinant uvrC plasmid and E. coli cells with pUVC1234 plasmids containing the endogenous uvrC operon (12). Because of the minute quantity of UvrC purified from these cells, we were unable to determine their activities. However, because even in cells producing minute amounts of UvrC proteins we were able to isolate UvrC that had an elution profile identical to that of UvrCI and UvrCII, it is likely that these two forms of UvrC proteins are the normal uvrC gene products and are not the results of overproduction.

We speculate that UvrCII is a major form existing in wild-type cells for two reasons. One is that UvrCII should be active in incision in vivo, because the cellular nucleotide/protein molar ratio is relatively large. The second reason is that because each cell has only <10 molecules of UvrC, and UvrCII does not form a complex with the free forms of the UvrA or UvrB protein, to account for the fast and efficient excision repair in vivo, the UvrC proteins must be concentrated in the genome area. The loose association of UvrCII to DNA would fulfill this critical requirement.


    ACKNOWLEDGEMENTS

We thank Dr. A. Sancar and Dr. R. Moses for providing uvrC-containing plasmids, Dr. A. Chen for determining pI, and Dr. S. Lloyd for determining the N-terminal amino acid sequence.


    FOOTNOTES

* This research was supported by Grants ES03124 and ES08389 from the United States Public Health Service.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.

Dagger To whom correspondence should be addressed. Tel.: 845-731-3585; Fax: 845-351-3492; E-mail: tang@env.med.nyu.edu.

Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M008538200


    ABBREVIATIONS

The abbreviations used are: ds, double-stranded; ss, single-stranded; RF, replicative form; PAGE, polyacrylamide gel electrophoresis; bp, base pair.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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