INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

DNA helicase-catalyzed unwinding of duplex DNA provides the single-stranded DNA (ssDNA) intermediates required for the reactions of DNA metabolism, including DNA replication, recombination, transcription, bacterial conjugation, and DNA repair (25, 26, 31, 32). Helicase enzymes use energy derived from nucleoside 5'-triphosphate (NTP) hydrolysis to catalyze the disruption of hydrogen bonds between the two strands in duplex DNA. The mechanism by which ATP hydrolysis is coupled with strand separation is not well understood. However, several models have been proposed and are currently being tested (27, 28, 48). An important element of these models is the requirement of multiple DNA binding sites for active translocation along DNA. For monomeric helicases, this requirement suggests the presence of multiple DNA binding sites on each individual protomer (36). In the case of oligomeric helicases (generally dimers or hexamers), individual ligand binding sites are on each monomer and oligomerization provides multiple DNA binding sites (27).

DNA helicases have been characterized in a variety of organisms, including bacteria, bacteriophages, viruses, and eukaryotes (for reviews see references 31 and 32). Eleven distinct helicases have been identified in Escherichia coli (30-32). The existence of multiple helicases within a single cell could reflect functional redundancy, either direct substitution of one helicase for another or an overlap in the biochemical pathways in which the proteins are involved. There is no evidence for the direct substitution of one helicase for another in a specific pathway, and at least in E. coli, each helicase appears to have a distinct biological role (31).

Alignments of the primary structures of helicases have been used to categorize helicases into several superfamilies (8-10, 17). Within superfamily I and II helicases, seven regions of sequence similarity, the so-called helicase motifs, have been identified. Sequence similarity among helicases is greatest within the motifs and tends to diverge outside these regions (8-10, 17). The conserved motifs are believed to have functional significance in the biochemistry of helicase-catalyzed unwinding. Indeed, this has been demonstrated for several different helicases in structure-function studies (2, 7, 11, 14, 15, 29, 41, 42, 50, 51). From these studies it has become clear that the helicase-associated motifs are required for the biological and biochemical functions of these proteins. The sequence conservation suggests that regions of a helicase outside the defined motifs may be responsible for dictating the biological specificity of a DNA helicase. Unique regions in each protein may direct participation in a particular biochemical pathway by guiding interactions with a specific DNA substrate or other proteins, or by directing its oligomerization for those proteins that form dimers or hexamers.

The biological specificity of DNA helicases has been well documented with the closely related DNA helicase II and Rep protein. DNA helicase II is required in methyl-directed mismatch repair (12, 13, 21, 37) and UvrABC-mediated excision repair (4, 18, 39, 44). Rep protein, which exhibits 40% identity with helicase II along its entire length and 90% identity within the helicase-associated motifs (9), cannot substitute for helicase II in either pathway. Conversely, Rep protein has a role in X174 DNA replication (6), and helicase II cannot substitute for Rep protein in this capacity. Presumably this high degree of specialization is due to specific protein-protein or protein-DNA interactions.

The amino acid sequences of Rep protein and helicase II diverge most dramatically in the C-terminal region of each protein, outside motif VI (9). The functional importance of the C terminus is evident for many proteins with helicase activity including the gp helicase-primase from bacteriophage P4 and the Rad25 helicase from yeast (40, 52). In addition, several mutations of the Werner's syndrome protein have been identified as C-terminal truncations outside the conserved helicase motifs (49).

A region near the C-terminal end of E. coli helicase II, and outside conserved motif VI, was defined as required for biological and biochemical function in assays using three mutants: UvrD107C (deletion of 107 C-terminal amino acids), UvrD102C, and UvrD40C. The mutant proteins UvrD107C and UvrD102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair, while UvrD40C protein fully restored activity in both genetic assays. The defect in UvrD102C was confirmed in vitro by its failure to bind ssDNA and a dramatically reduced ssDNA-stimulated ATPase activity. UvrD40C was essentially indistinguishable from wild-type UvrD in biochemical activity assays. The results suggest a role for a region near the C terminus, residues 618 to 680, of helicase II in DNA binding.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains. E. coli BL21(DE3) (F ompT[lon] hsdS_B(r_Bm_B) gal dcm DE3) was from Novagen, Inc. E. coli GE1752 (metE::cam) was obtained from G. Weinstock. BL21(DE3) uvrD and GE1752 uvrD were constructed previously in this laboratory (7).

DNA and nucleotides. pET11d and pLysS were from Novagen, Inc. pMal-C2 and pTYB4 were from New England Biolabs, Inc. (NEB). M13mp7 ssDNA was prepared as described elsewhere (24). Unlabeled nucleotides were from U.S. Biochemical Corp. Radioactively labeled nucleotides were from Amersham Corp. pET11d-UvrD was constructed previously in this laboratory (7).

Mutagenesis. Plasmids capable of expressing UvrD107C, UvrD102C, maltose-binding protein (MBP)-UvrD102C, and intein-UvrD102C were constructed by PCR with Vent polymerase and appropriate primers. pET11d-UvrD was used as a template for amplification. UvrD107C and UvrD102C were amplified by using a primer (5'-GGAATTGTGAGCGGATAACAATTCCCC-3') that annealed upstream of the uvrD coding sequence and primer 107C (5'CGGAGATCTCATTAGGTCAGCGTCAGTTTCTGC-3') or primer 102C (5'-GCCCAGATCTAAGCTTATTAGCGGGTTTCCGCGTAGG-3'). Primer 107C altered codon 614 of helicase II from TAC (tyrosine) to TAA (stop) and codon 615 GCG (alanine) to TGA (stop). Primer 102C changed codon 619 from CGT (arginine) to TAA (stop) and codon 620 from CTG (leucine) to TAA (stop). Following amplification, the DNA was cleaved with restriction enzymes NcoI and BglII. These restriction sites were engineered into the primers. Gel-purified DNA fragments were subcloned into the pET11d vector that had been digested with NcoI and BamHI.

uvrD40C

uvrD102C

Enzymes. Restriction endonucleases, DNA polymerase I (large fragment), phage T4 polynucleotide kinase, and Vent DNA polymerase were from NEB and used as recommended by the supplier. Phage T4 DNA ligase was from Boehringer Mannheim and used as advised.

1

1

Genetic assays. All genetic assays were performed with BL21(DE3)/pLysS or BL21(DE3)uvrD/pLysS. UV irradiation survival assays were as described previously (1). To determine the spontaneous mutation rate, 11 independent isolates of each indicated cell strain were grown in LB-glucose (0.4%) medium under antibiotic selection at 37°C. LB-glucose medium was necessary to alleviate problems associated with plasmid loss in these experiments. Serial dilutions of each saturated culture were made in M9 minimal medium salts. Cell titer was determined by plating appropriate dilutions on LB-agar or LB-agar plus ampicillin (200 µg/ml). Dilutions were plated on LB-agar plus rifampin (100 µg/ml) to ascertain the number of spontaneously arising rifampin-resistant colonies. Plates were incubated at 37°C for at least 24 h. Colonies were counted, and the spontaneous mutation rate was calculated for each strain by the method of the median as described elsewhere (23).

DNA binding assays. DNA binding was evaluated by measuring the retention of a [³²P]DNA ligand on nitrocellulose filters as described previously (15, 34). Reaction mixtures (20 µl) contained 25 mM Tris-HCl (pH 7.5), 3 mM MgCl₂, 20 mM NaCl, 3 mM adenosine 5'-O-(thiotriphosphate) (ATPS), 5 mM 2-mercaptoethanol, 50 µg of bovine serum albumin per ml, and a ³²P-labeled 90-base oligonucleotide at approximately 0.9 µM DNA phosphate (0.01 µM molecules) (6.74 × 10⁸ cpm µmol¹). Reactions were initiated by adding the indicated amount of protein, and reaction mixtures were incubated at 37°C for 10 min. After incubation, 1 ml of prewarmed (37°C) reaction buffer was added. The entire mixture was filtered on a HAWP (Millipore) filter at a flow rate of about 4 ml/min. Filters were washed two times with 1 ml of reaction buffer and dried for liquid scintillation counting. Background values were typically less than 1% of the total radioactivity.

ATPase assays. Measurements of DNA-stimulated ATP hydrolysis were completed as described elsewhere (33). Reaction mixtures for evaluating the k_cat and K_m contained 25 mM Tris-HCl (pH 7.5), 3 mM MgCl₂, 20 mM NaCl, 5 mM 2-mercaptoethanol, and M13mp7 ssDNA (30 µM nucleotide phosphate). In k_cat assays, 30- or 60-µl (for duplicate trials) reaction mixtures were prewarmed at 37°C and initiated by the addition of protein. Dilutions of protein were made in storage buffer. [³H]ATP was at a concentration of 0.8 mM. Samples (5 µl) were removed at 2-min intervals from reaction mixtures containing UvrD or UvrD40C. Samples (5 µl) were removed at 5, 10, 20, 40, and 60 min from reaction mixtures containing UvrD102C. Reaction tubes without protein were incubated in the same manner and used as controls. Each sample was quenched with 5 µl of stop solution (33 mM EDTA, 7 mM ADP, 7 mM ATP). Quenched reactions were processed as described elsewhere (33). The UvrD102C-catalyzed ATP hydrolysis rate remained in the linear range throughout the entire time course (60 min) and thus reflects an initial rate. Thirty-minute incubations produced the same calculated k_cat values (data not shown).

Helicase assays. The DNA unwinding activities of UvrD, UvrD40C, and MBP-UvrD102C were compared by using a 238-bp blunt duplex substrate. The DNA unwinding activities of UvrD and MBP-UvrD102C were also evaluated with a 92-bp partial duplex. The 92-bp partial duplex was prepared as described elsewhere (2, 35). Reaction mixtures contained approximately 1.3 µM nucleotide phosphate.

Proteolytic digestion. Limited digestion of UvrD and UvrD102C was performed with -chymotrypsin (Sigma). Reaction mixtures (25 µl) contained 1.5 µM UvrD or 1.6 µM UvrD102C (purified from an intein fusion). UvrD-containing reaction mixtures were incubated with 4.3 ng of chymotrypsin, and UvrD102C-containing reaction mixtures were incubated with 5.6 ng. Tubes were prewarmed for 30 s before reactions were initiated. Reactions were initiated by the addition of chymotrypsin, the mixtures were incubated for 4 min at 37°C, and the reactions were stopped by addition of 25 µl of gel loading buffer (250 mM Tris-HCl [pH 6.8], 3.4% SDS, 1.1 M 2-mercaptoethanol, 20% glycerol, 0.01% bromophenol blue) and boiling. Products were resolved on a 9.6% polyacrylamide gel (32:1 cross-linking ratio) run in the presence of 0.1% SDS. Proteins were visualized with Coomassie brilliant blue R-250 (Sigma).

Circular dichroism. Samples were prepared for circular dichroism measurements by extensive dialysis into CD buffer (see above). Protein samples were filtered (0.2 µm) to remove any aggregated material. Measurements were made with a Jasco J600 spectropolarimeter. Wavelength scans of UvrD (4.6 µM), UvrD40C (6.9 µM), and UvrD102C (6.5 µM) were from 200 to 260 nm at 20°C. The cuvette used was a 0.1-cm quartz cell. Scans of protein material were normalized to scans of the buffer without protein.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

The significance of the C terminus of helicase II (UvrD), a region outside the conserved helicase-associated motifs, was investigated by constructing and evaluating the biological and biochemical function of three truncation mutants: UvrD40C, UvrD102C, and UvrD107C. These mutants were constructed as described in Materials and Methods and are shown in Fig. 1. A protein essentially identical to UvrD102C was the subject of a previous study (5). UvrD40C was constructed because this truncation was significantly further away from the end of motif VI than the other two mutant proteins.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Structures and nomenclature of helicase II (UvrD) C-terminal deletion mutants. Approximate positions of six conserved helicase motifs are shown for UvrD (motif Ia is not shown). The sequence of motif VI, as described by Gorbalenya et al. (10) is in boldface. The arrow points to a trypsin cleavage site previously described by Chao and Lohman (5). An asterisk denotes the end of each polypeptide. Amino acid residue numbers are indicated above the sequence for wild-type UvrD.

The ability of each truncation mutant to complement a deletion of the uvrD gene in genetic assays was evaluated. In addition, UvrD40C, UvrD102C, and MBP-UvrD102C were purified and biochemically characterized. The MBP-UvrD102C and the intein-UvrD102C construct were made to facilitate the purification of UvrD102C (see Materials and Methods). Previous studies have shown that an MBP fusion to wild-type UvrD results in a protein with wild-type activity (data not shown). UvrD102C purified from the intein fusion had a K_m for ATP hydrolysis similar to that of UvrD102C purified from pET11d (data not shown).

Genetic characterization of UvrD40C and UvrD102C. E. coli DNA helicase II has an essential role in methyl-directed mismatch repair and UvrABC-directed nucleotide excision repair (4, 18, 21). The ability of each truncation mutant to function in these pathways was evaluated by genetic complementation tests using strains lacking the wild-type uvrD gene. Both complementation assays required the use of bacterial strains containing the DE3 prophage that encodes T7 RNA polymerase. In each case, the mutant gene was introduced on an expression plasmid that utilized phage T7 transcription-translation initiation signals. For reasons that are not understood, and unlike other mutants and wild-type UvrD, expression of UvrD102C and UvrD107C was not detectable in the absence of the prophage (data not shown). Thus, we assume that expression of these plasmid-encoded truncation mutants is driven by T7 RNA polymerase. It should be noted that induction of the expression of T7 RNA polymerase by the addition of IPTG was not required to observe expression of these mutant uvrD alleles. The level of expression of UvrD, UvrD102C and UvrD40C in BL21(DE3)/pLysS cells was somewhat greater (less than 10-fold) than chromosomal levels of UvrD expression, as judged by Western blot analysis of cell lysates (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   UV survival of BL21(DE3)/pLysS cells and derivatives. E. coli BL21(DE3)/pLysS (uvrD+) (), BL21(DE3)uvrD/pLysS (uvrD) (), BL21(DE3)uvrD/pLysS/pET11d-UvrD (uvrD+) (), BL21(DE3)uvrD/pLysS/pET11d-UvrD40C (uvrD40C) (), and BL21(DE3)uvrD/pLysS/pET11d-UvrD102C (uvrD102C) () were exposed to increasing doses of UV light as indicated; 100% survival was defined as the number of colonies present in an unirradiated sample. Data are averages of three individual experiments.

Biochemical characterization of UvrD102C and UvrD40C. UvrD40C was purified by using a previously published helicase II purification procedure (43), with one exception. The protein failed to bind an ssDNA-cellulose column equilibrated at 0.2 M NaCl. Purification of this protein required the loading of the ssDNA-cellulose at a lower salt concentration (0.1 M NaCl).

View larger version (31K): [in this window] [in a new window]   FIG. 3.   SDS-polyacrylamide gel analysis of purified proteins. Purified proteins were resolved on a 9.6% polyacrylamide gel in the presence of SDS and stained with Coomassie blue. Lane 2, 1.2 µg of UvrD; lane 3, 1.1 µg of UvrD40C; lane 4, 1 µg of UvrD102C; lane 5, 1.2 µg of MBP-UvrD102C. Molecular weight standards (lane 1) were rabbit muscle phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg white ovalbumin (45.0 kDa), and bovine carbonic anhydrase (31.0 kDa).

(i) DNA binding. The behavior of UvrD40C and UvrD102C on ssDNA-cellulose columns during purification suggested possible defects in the interaction of these proteins with DNA. The binding of UvrD102C and UvrD40C to ssDNA was directly evaluated by using a nitrocellulose filter binding assay and a [³²P]DNA ligand (90-mer oligonucleotide). DNA concentration was held constant while protein concentration was varied as indicated. Results of DNA binding experiments performed in the presence of a poorly hydrolyzed ATP analog, ATPS, are shown in Fig. 4. UvrD40C behaved like wild-type helicase II under these conditions. UvrD102C, however, was markedly defective for binding to ssDNA.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   DNA binding using UvrD, UvrD40C, and UvrD102C. Nitrocellulose filter binding experiments with UvrD (), UvrD40C (), and UvrD102C () were performed as described in Materials and Methods, using the indicated concentration of each purified protein. Data represent averages of several trials. Error bars show the standard error about the mean.

(ii) ATPase and helicase activities. To better define the enzymatic properties of UvrD102C and UvrD40C, both proteins were assayed for ATPase and helicase activities. The k_cat values for ssDNA-stimulated ATP hydrolysis catalyzed by UvrD and UvrD40C were essentially the same. UvrD102C was significantly deficient in DNA-stimulated ATP hydrolysis (Table 2). The k_cat for this protein was 0.01% that of the wild-type protein.

View this table: [in this window] [in a new window]   TABLE 2.   DNA-stimulated ATP hydrolysis by UvrD, UvrD40C, and UvrD102Ca

Protein conformation studies. The ability of UvrD102C to interact with ATP indicates that loss of the 102 C-terminal amino acids has not caused the disruption of the global fold of the protein. The possibility that UvrD102C may be defective as a helicase due to an alteration of the overall or local conformation or due to an inability of this protein to make some of the necessary conformational changes believed to be associated with ATP hydrolysis was further addressed by the use of circular dichroism measurements and limited proteolytic cleavage.

View larger version (28K): [in this window] [in a new window]   FIG. 5.   Limited proteolysis of UvrD and UvrD102C with -chymotrypsin. (A) UvrD (1.5 µM) and UvrD102C (1.6 µM) were subjected to limited proteolysis as described in Materials and Methods (lanes 2 and 4, respectively). Lanes 1 and 3 contain UvrD and UvrD102C in the absence of chymotrypsin. This gel was stained with Coomassie blue. Molecular weight standards were the same as in Fig. 3. Arrows indicate full-length UvrD102C and the 42- and 29-kDa proteolytic fragments. (B) UvrD (lanes 1 to 3; 0.6 µM) and UvrD102C (lanes 4 to 6; 0.6 µM) were incubated in the absence (lanes 1 and 4) or presence (lanes 2, 3, 5, and 6) of 10 ng of chymotrypsin. Reaction mixtures were preincubated in the presence (lanes 2 and 5) or absence (lanes 3 and 6) of 3.2 mM ATP and MgCl2. Previous studies have shown that chymotrypsin is not inhibited by the presence of either MgCl2 or ATP (2, 16). The products of digestion were resolved on a 9.6% polyacrylamide gel in the presence of SDS. Products were visualized by Western blotting with antibodies to wild-type helicase II. Molecular weight standards were prestained broad-range markers (Bio-Rad).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The results presented here indicate that a region near the C-terminal end of DNA helicase II is necessary for in vivo function of the protein in both the excision repair and methyl-directed mismatch repair pathways. In vivo defects correlate with in vitro defects including a markedly impaired interaction with DNA and nearly undetectable ATPase and helicase activities. This result was, perhaps, unexpected since this region of the protein is located outside of the conserved helicase motifs. Amino acids associated with these motifs are assumed, and in many cases known, to be essential for the biochemical activities of this class of enzymes. However, the results presented here indicate that regions outside of the identified motifs also play significant roles in the biochemical mechanism of DNA unwinding.

It was previously reported that the C terminus was dispensable for the unwinding and ATPase activities of helicase II (5). In those experiments helicase II was digested with trypsin to produce a 72-kDa polypeptide lacking the C-terminal end of the protein (see Fig. 1 for the location of the trypsin cleavage site). The results reported here with UvrD102C reveal a very significant defect in the ATPase/helicase reaction catalyzed by this protein. A possible explanation for this discrepancy centers on the fact that the 72-kDa polypeptide was not purified in the previous study (5). The 10-kDa C-terminal polypeptide may have remained associated with the larger fragment, masking the defect caused by the truncation. It is also possible that under the conditions chosen for the trypsin reaction, UvrD had oligomerized further aiding in the association of the C-terminal peptide fragment with the remainder of the protein. There is precedence for the proteolytic fragments of UvrD remaining associated with each other. Chao and Lohman (5) report that chymotrypsin cleavage products (29 and 53 kDa) copurify under nondenaturing conditions, suggesting a physical association between these cleavage products. Moreover, these associated chymotrypsin cleavage products were able to bind ssDNA despite the fact that the cleavage site is located in the middle of motif III, which has been implicated in DNA binding (2, 20). Alternatively, there is the potential for contamination by full-length helicase II in the proteolytically digested preparations. The genetically altered UvrD truncations used in these studies were not subject to any of these potential problems.

Nitrocellulose filter binding experiments clearly demonstrate the failure of UvrD102C to bind ssDNA. These results were consistent with the failure of this protein to bind ssDNA-cellulose during purification. This defect is sufficient to explain the genetic resultsUvrD102C is unable to function in excision repair or methyl-directed mismatch repair because it cannot bind the necessary DNA substrates. UvrD40C appeared slightly less efficient in DNA binding, suggesting that this truncation was beginning to affect a region of the protein that was important for DNA binding. However, this very slight DNA binding defect did not impair in vivo function of this enzyme. It should be noted here that UvrD40C has been shown to fail to dimerize and that dimerization is not essential for the in vitro and in vivo activities of helicase II (36). In other helicases, for example, gp and eIF-4B, regions at or near the C terminus have been shown to have a role in DNA and RNA binding (38, 52).

An obvious concern when one is evaluating the biochemical activity of a truncated protein is disturbance of the conformation of the protein. Several lines of evidence suggest that UvrD102C is properly folded. First, the protein was successfully overexpressed and was soluble in whole-cell lysates. Misfolded proteins are often quickly degraded or insoluble in the cell. Second, based on previous limited proteolysis experiments (5), UvrD102C represents an independently folding domain. Data from circular dichroism measurements demonstrate that this protein has significant secondary structure. Third, additional proteolysis experiments indicate that this protein is folded correctly, as evidenced by the presence of the appropriate chymotrypsin cleavage site (Fig. 5). Finally, UvrD102C interacts with nucleotide in the same fashion as wild-type helicase II. The K_m values for UvrD and UvrD102C were essentially identical. Moreover, UvrD102C and UvrD are both protected from digestion with chymotrypsin in the presence of ATP (Fig. 5).

Even though UvrD102C had a K_m for ATP similar to that of UvrD, it had a significantly lower k_cat value for ATP hydrolysis than UvrD. Not surprisingly, this reaction was not stimulated by the addition of ssDNA. It should be noted that the k_cat value was lower than that measured for DNA-independent ATP hydrolysis catalyzed by wild-type UvrD (0.4 s¹). The reason for this difference is not clear. If the region between amino acids 618 and 680 was simply required for ssDNA binding, then one might expect UvrD102C to have a DNA-independent hydrolysis activity equivalent to that of wild-type UvrD. However, the mechanism of DNA-independent ATP hydrolysis is not well characterized. Evidence suggests that ATP may bind in the ssDNA binding region, albeit with low affinity. Substrate inhibition of ssDNA-stimulated ATP hydrolysis at high ATP concentrations has been observed for the wild-type enzyme. Kinetic studies suggest this is the result of competition for the DNA binding site (42a). Therefore, in the case of the wild-type enzyme, ATP could bind in the DNA binding pocket, when present at sufficiently high concentration, and act to stimulate ssDNA-independent hydrolysis by acting as an allosteric effector. Due to the absence of this region in UvrD102C, this allosteric stimulation by ATP would presumably not occur with UvrD102C.

Other explanations are also possible. This region may be directly involved in the ATP hydrolysis reaction. The ssDNA binding regions are located near the ATP binding pocket in the structure of Rep protein (20). Another possibility is that the C-terminal amino acids serve as a bridge of communication between domains of helicase II. The individual domains of helicase II might have independent roles in the cycle of ATP binding, hydrolysis, and product release. Coordination of the activities of these domains could be required for DNA-independent ATP hydrolysis. Finally, the C terminus may function as a switch that regulates the cycling of conformational states in order to couple ATP hydrolysis to the disruption of hydrogen bonds. Although the protein is able to change conformation in response to ATP binding, perhaps without the C-terminal region the protein cannot continuously cycle, preventing efficient hydrolysis of ATP.

The data presented here clearly implicate residues 618 to 680 as part of helicase II required for a stable interaction with ssDNA. It is known, from site-directed mutagenesis studies (2, 16) and from crystal structures of the related Rep and PcrA proteins (20, 47), that other regions of the protein also have roles in DNA binding. In the Rep cocrystal with bound dT(pT)₁₅ several amino acids that contact bound ssDNA were identified. Some of these residues lie in motifs Ia, III, and V, between motifs IV and V, and between motifs V and VI. In this structural model, the DNA molecule was located in a large cleft that runs between the subdomains of the protein (20). The DNA contact regions identified in the Rep crystal structure were consistent with several of the putative DNA binding regions proposed for PcrA, based on its structural similarity with RecA (47). Consistent with the idea of multiple DNA binding regions, the C terminus of helicase II may serve as an additional portion of the protein with a role in DNA binding. The C terminus is absent in the solved structures of PcrA and Rep protein (it was too disordered to be resolved). This flexible region (residues 618 to 680) could be seen as overlying the central pocket of the protein, where ssDNA is thought to bind, serving as a type of clamp (20, 47). This clamp could stabilize the interaction between helicase II and ssDNA either by physically holding the DNA in place or by locking the protein in a DNA-bound conformational state. The multiple regions of the protein involved in DNA binding may or may not be independently regulated. There is not enough known to differentiate between these possibilities. Ultimately, the role of the C terminus will only be fully understood once a structure of helicase II is determined with resolution of this C-terminal region.

All of the data presented establish that while the helicase motifs may be associated with all helicases, and are required for unwinding activity, these motifs alone do not define a functional helicase domain for UvrD. Moreover, there are several known examples of putative helicases that contain all the helicase-associated motifs but fail to catalyze a helicase reaction in vitro. These include the E. coli and eukaryotic transcription repair coupling factor (45, 46) and the yeast Rad5 protein (19). Thus, precisely what determines a functional helicase is not yet known with certainty. While helicases contain conserved motifs that are critical for the biochemical mechanism of unwinding, these motifs are not the only regions of these proteins required for enzyme activity. Thus, regions lying outside of the motifs may tailor these proteins to specific biochemical roles.