The regulatory particle of the Saccharomyces cerevisiae proteasome

The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.     

Molecular epidemiology of human polyomavirus JC in the Biaka Pygmies and Bantu of Central Africa

The proteasome consists of a 20S proteolytic core particle (CP) and a 19S regulatory particle (RP), which selects ubiquitinated substrates for translocation into the CP. An eight-subunit subcomplex of the RP, the lid, can be dissociated from proteasomes prepared from a deletion mutant for Rpn10, an RP subunit. A second subcomplex, the base, contains all six proteasomal ATPases and links the RP to the CP. The base is sufficient to activate the CP for degradation of peptides or a nonubiquitinated protein, whereas the lid is required for ubiquitin-dependent degradation. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The lid subunits share sequence motifs with components of the COP9/signalosome complex and eIF3, suggesting that these functionally diverse particles have a common evolutionary ancestry.     

The role of the ClpA chaperone in proteolysis by ClpAP

ClpA, a member of the Clp/Hsp100 family of ATPases, is a molecular chaperone and, in combination with a proteolytic component ClpP, participates in ATP-dependent proteolysis. We investigated the role of ClpA in protein degradation by ClpAP by dissociating the reaction into several discrete steps. In the assembly step, ClpA-ClpP-substrate complexes assemble either by ClpA-substrate complexes interacting with ClpP or by ClpA-ClpP complexes interacting with substrate; ClpP in the absence of ClpA is unable to bind substrates. Assembly requires ATP binding but not hydrolysis. We discovered that ClpA translocates substrates from their binding sites on ClpA to ClpP. The translocation step specifically requires ATP; nonhydrolyzable ATP analogs are ineffective. Only proteins that are degraded by ClpAP are translocated. Characterization of the degradation step showed that substrates can be degraded in a single round of ClpA-ClpP-substrate binding followed by ATP hydrolysis. The products generated are indistinguishable from steady-state products. Taken together, our results suggest that ClpA, through its interaction with both the substrate and ClpP, acts as a gatekeeper, actively translocating specific substrates into the proteolytic chamber of ClpP where degradation occurs. As multicomponent ATP-dependent proteases are widespread in nature and share structural similarities, these findings may provide a general mechanism for regulation of substrate import into the proteolytic chamber.     

Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.     

Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity

We have identified mutations in Raf-1 that increase binding to Ras. The mutations were identified making use of three mutant forms of Ras that have reduced Raf-1 binding (Winkler, D. G., Johnson, J. C., Cooper, J. A., and Vojtek, A. B. (1997) J. Biol. Chem. 272, 24402-24409). One mutation in Raf-1, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation. Missense substitutions of residues 143 and 144 in the Raf-1 cysteine-rich domain were isolated multiple times. These Raf-1 mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras. Each was slightly activated relative to wild-type Raf-1 in a transformation assay. In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes. Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-1. We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-1 is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-1 and increasing Raf-1 activity.     

Pain and discomfort associated with common hospital procedures and experiences

BACKGROUND: Protein kinases play a central role in controlling diverse signal transduction pathways in all cells. The identification of the direct cellular substrates of individual protein kinases remains the key challenge in the field. RESULTS: We describe the protein engineering of v-Src to produce a kinase which preferentially uses an ATP analog, N6-(benzyl) ATP, as a substrate, rather than the natural v-Src substrate, ATP. The sidechain of a single residue (Ile338) controls specificity for N6-substituted ATP analogs in the binding pocket of v-Src. Elimination of this sidechain by mutation to glycine produces a v-Src kinase which preferentially utilizes N6-(benzyl) ATP as a phosphodonor substrate. Our engineering strategy is generally applicable to the Src family kinases: mutation of the corresponding residue (Thr339 to glycine) in the Fyn kinase confers specificity for N6-(benzyl) ATP on Fyn. CONCLUSIONS: The v-Src tyrosine kinase has been engineered to exhibit specificity for an unnatural ATP analog, N6-(benzyl) ATP, even in a cellular context where high concentrations of natural ATP are present (1-5 mM), where preferential use of the ATP analog by the mutant kinase is essential. The mutant v-Src transfers phosphate more efficiently with the designed unnatural analog than with ATP. As the identical mutation in the Src-family kinase Fyn confers on Fyn the ability to recognize the same unnatural ATP analog, our strategy is likely to be generally applicable to other protein kinases and may help to identify the direct targets of specific kinases.     

Directed evolution of an aspartate aminotransferase with new substrate specificities

The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuffling and selection in an auxotrophic Escherichia coli strain. After five rounds of selection, one of the evolved mutants showed a 10(5)-fold increase in the catalytic efficiency (kcat/Km) for beta-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme. The mutant had 13 amino acid substitutions, 6 of which contributed 80-90% to the total effect. Five of these six substitutions were conserved among the five mutants that showed the highest activity for beta-branched substrates. Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult. The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied.     

Visualization of the dopamine transporter in the human brain postmortem with the new selective ligand [125I]PE2I

In a genetic screen aimed at the identification of trans-acting factors involved in mRNA 3'-end processing of budding yeast, we have previously isolated two temperature-sensitive mutants with an apparent defect in the 3'-end formation of a plasmid-derived pre-mRNA. Surprisingly, both mutants were rescued by the essential gene ESS1/PTF1 that encoded a putative peptidylprolyl-cis/trans-isomerase (PPIase) (Hani, J., Stumpf, G., and Domdey, H. (1995) FEBS Lett. 365, 198-202). Such enzymes, which catalyze the cis/trans-interconversion of peptide bonds N-terminal of prolines, are suggested to play a role in protein folding or trafficking. Here we report that Ptf1p shows PPIase activity in vitro, displaying an unusual substrate specificity for peptides with phosphorylated serine and threonine residues preceding proline. Both mutations were found to result in amino acid substitutions of highly conserved residues within the PPIase domain, causing a marked decrease in PPIase activity of the mutant enzymes. Our results are suggestive of a so far unknown involvement of a PPIase in mRNA 3'-end formation in Saccharomyces cerevisiae.     

Saccharomyces cerevisiae Msh2p and Msh6p ATPase activities are both required during mismatch repair

In the Saccharomyces cerevisiae Msh2p-Msh6p complex, mutations that were predicted to disrupt ATP binding, ATP hydrolysis, or both activities in each subunit were created. Mutations in either subunit resulted in a mismatch repair defect, and overexpression of either mutant subunit in a wild-type strain resulted in a dominant negative phenotype. Msh2p-Msh6p complexes bearing one or both mutant subunits were analyzed for binding to DNA containing base pair mismatches. None of the mutant complexes displayed a significant defect in mismatch binding; however, unlike wild-type protein, all mutant combinations continued to display mismatch binding specificity in the presence of ATP and did not display ATP-dependent conformational changes as measured by limited trypsin protease digestion. Both wild-type complex and complexes defective in the Msh2p ATPase displayed ATPase activities that were modulated by mismatch and homoduplex DNA substrates. Complexes defective in the Msh6p ATPase, however, displayed weak ATPase activities that were unaffected by the presence of DNA substrate. The results from these studies suggest that the Msh2p and Msh6p subunits of the Msh2p-Msh6p complex play important and coordinated roles in postmismatch recognition steps that involve ATP hydrolysis. Furthermore, our data support a model whereby Msh6p uses its ATP binding or hydrolysis activity to coordinate mismatch binding with additional mismatch repair components.     

Identification and functional analysis of sulfonylurea receptor 1 variants in Japanese patients with NIDDM

The sulfonylurea receptor 1 (SUR1) is an essential regulatory subunit of the beta-cell ATP-sensitive K+ channel (K[ATP]). The possible role of SUR1 gene mutation(s) in the development of NIDDM remains controversial as both a positive association and negative linkage results have been reported. Therefore, we examined the SUR1 gene at the single nucleotide level with single strand conformation polymorphism analysis in 100 Japanese NIDDM patients. We identified a total of five amino acid substitutions and 17 silent mutations by examining all 39 exons of this gene. Two rare novel mutations, D811N in exon 20 and R835C in exon 21, were identified in the first nucleotide-binding fold (NBF), a functionally important region of SUR1, in one patient each, both heterozygotes. To analyze possible functional alterations, we reconstituted the mutant K(ATP) by coexpressing beta-cell inward rectifier (BIR) (Kir 6.2), a channel subunit of K(ATP), and mutant SUR1 in HEK293T and COS-7 cells. As demonstrated by the patch clamp technique and rubidium (Rb+) efflux studies, neither mutation alters the properties of channel activities. Two other rare missense mutations, R275Q in exon 6 and V560M in exon 12, were also identified. The R275Q substitution was not found in 67 control subjects, and V560M was present in three control subjects. Neither of these substitutions appeared to cosegregate with NIDDM in the probands' families. A previously reported S1370A substitution located in the second NBF was also common in the Japanese subjects (allelic frequency 0.37), and was found at an equal frequency in nondiabetic control subjects. In conclusion, SUR1 mutations impairing K(ATP) function do not appear to be major determinants of NIDDM susceptibility in Japanese.     

The ATP-binding cassette (ABC) transporter for maltose/maltodextrins of Salmonella typhimurium. Characterization of the ATPase activity associated with the purified MalK subunit

The ATPase activity associated with the purified MalK subunit of the maltose transport complex of Salmonella typhimurium, a bacterial ATP-Binding Cassette (ABC) transporter (Walter, C., H?ner zu Bentrup, K., and Schneider, E. (1992) J. Biol. Chem. 267, 8863-8869), was characterized in detail. The analysis of the kinetics of ATP hydrolysis yielded a Km value of 70 +/- 4 microM and a Vmax of 1.3 +/- 0.3 mumol/min/mg of protein. Both GTP and CTP also served as substrates. While MalK exhibited nearly the same affinity for GTP as for ATP, the Michaelis constant for CTP as a substrate was much higher. ATP hydrolysis was strongly dependent on the presence of Mg2+ ions. Mn2+ at low concentrations, but neither Ca2+ nor Zn2+ partially substituted for Mg2+. The ATPase activity was optimal at slightly alkaline pH and was stimulated in the presence of both glycerol (7.5%) and dimethyl sulfoxide (Me2SO) (5%). ADP and the non-cleavable substrate analog ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) were identified as competitive inhibitors. The MalK-ATPase was resistant to specific inhibitors of F-, P-, and V-type ATPases, such as dicyclohexylcarbodiimide, azide, vanadate, or bafilomycin A1. In contrast, micromolar concentrations of the sulfhydryl reagent N-ethylmaleimide strongly inhibited the enzymatic activity. This inhibition was blocked in the presence of ATP. These results suggest that the intrinsic ATPase activity of purified MalK can be clearly distinguished from other ATP-hydrolyzing enzymes, e.g. ion-translocating ATPases.     

Functional role of the N-terminal region of the Lon protease from Mycobacterium smegmatis

Lon protease homologues contain a poorly conserved N-terminal region of variable length. To better understand the role of the N-terminal region of Lon in the complicated reaction cycle of ATP-dependent protein degradation, we expressed and characterized mutants of the Lon protease from Mycobacterium smegmatis (Ms-Lon) lacking 90, 225, and 277 N-terminal residues (N-G91, N-E226, and N-I278, respectively). N-I278 displayed neither peptidase nor ATPase activity despite the fact that it was stable and soluble in vivo, had a near-wild-type CD spectrum, and the deleted residues included neither the catalytic nucleophile for peptide bond hydrolysis (S675) nor the ATP binding regions. N-G91 and N-E226 retained peptidase activities against small unstructured peptides that were stimulated, to near-wild-type levels, by the Ms-Lon substrate protein alpha-casein. By contrast, N-G91 and N-E226 retained basal ATPase activities, but these activities were only stimulated weakly by alpha-casein. Ms-Lon, N-E226, and N-G91 all exhibited low-level peptidase activity in assays containing nonhydrolyzed nucleotide analogues. However, these peptidase activities were stimulated strongly by alpha-casein in the case of Ms-Lon but weakly by alpha-casein in the cases of N-G91 and N-E226. Strikingly, despite the near-wild-type peptidase activities of N-G91 and N-E226, both were severely impaired in their degradation of the Ms-Lon protein substrates alpha-casein in vitro and RcsA in vivo. Overall, N-G91 and N-E226 displayed catalytic properties similar to Escherichia coli Lon (Ec-Lon) in the presence of the PinA inhibitor, suggesting that PinA inhibits Ec-Lon protease by inhibiting the function of Ec-Lon's N-terminal region. In vivo protease assays further revealed that, in contrast to the inactive Ms-Lon point mutant S675A, N-G91 and N-E226 did not reduce the cellular activity of RcsA. This same defect was observed previously for Ms-Lons with multiple mutations in their peptidase active sites. We conclude that proteolytically inactive mutants of Ms-Lon retain the ability to reduce the cellular activity of RcsA but that both the N-terminal region and the peptidase active site region of Ms-Lon are required for this activity of wild-type Ms-Lon. The inabilities of N-G91 and N-E226 to degrade larger protein substrates and to reduce the cellular activity of RcsA were not the result of drastic alterations in their quaternary structures. Gel filtration profiles of N-G91 and N-E226 revealed that each was primarily tetrameric, with an increased percentage of dimeric species and a decreased percentage of trimeric species relative to Ms-Lon. The observed shifts in the dimer/trimer ratios of the N-terminal truncation mutants suggest that the Ms-Lon tetramer contains two types of subunit-subunit interactions.     

The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing

The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.     

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.     

Investigation of the relationship between farrowing environment, sex steroid concentrations and maternal aggression in gilts

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac alpha1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the alpha1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the alpha1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells.     

Ste6p mutants defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER quality control pathway in Saccharomyces cerevisiae

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6-166p, is highly unstable. We show that its degradation involves the ubiquitin-proteasome system, as indicated by its in vivo stabilization in certain ubiquitin-proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6-13p and Ste6-90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.     

Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone

Hsp70 chaperones assist protein folding by ATP-controlled cycles of substrate binding and release. ATP hydrolysis is the rate-limiting step of the ATPase cycle that causes locking in of substrates into the substrate-binding cavity of Hsp70. This key step is strongly stimulated by DnaJ cochaperones. We show for the Escherichia coli Hsp70 homolog, DnaK, that stimulation by DnaJ requires the linked ATPase and substrate-binding domains of DnaK. Functional interaction with DnaJ is affected by mutations in an exposed channel located in the ATPase domain of DnaK. It is proposed that binding to this channel, possibly involving the J-domain, allows DnaJ to couple substrate binding with ATP hydrolysis by DnaK. Evolutionary conservation of the channel and the J-domain suggests conservation of the mechanism of action of DnaJ proteins.     

Helicase motifs V and VI of the Escherichia coli UvrB protein of the UvrABC endonuclease are essential for the formation of the preincision complex

The UvrB protein is a subunit of the UvrABC endonuclease which is involved in the repair of a large variety of DNA lesions. We have 91 isolated random uvrB mutants which are impaired in the repair of UV-damage in vivo. These mutants were classified on the basis of the ability to form normal levels of protein and the position of the mutations in the gene. The amino acid substitutions in the N-terminal part or in the C-terminal part of the UvrB protein are exclusively found in the conserved boxes of the so-called "helicase motifs" present in these parts of the protein, indicating that these motifs are essential for UvrB function. The proteins of four C-terminal mutants were purified: two mutants in motif V (E514K and G509S), one mutant in motif VI (R544H) and a double mutant in both motifs (E514K + R541H). In vitro experiments with these mutant proteins show that the helicase motifs V and VI are involved in the induction of ATP hydrolysis in the presence of (damaged) DNA and in the strand-displacement activity of the UvrA2B complex as is observed in a helicase assay. Furthermore, our results suggest that this strand-displacement activity is correlated to a local unwinding, which seems to be used to form the UvrB-DNA preincision complex.