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• W2000675871 abstract "UvrB plays a major role in recognition and processing of DNA lesions during nucleotide excision repair. The crystal structure of UvrB revealed a similar fold as found in monomeric DNA helicases. Homology modeling suggested that the β-hairpin motif of UvrB might be involved in DNA binding (Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., and Kisker, C. (1999) EMBO J.18, 6899–6907). To determine a role of the β-hairpin ofBacillus caldotenax UvrB, we have constructed a deletion mutant, Δβh UvrB, which lacks residues Gln-97–Asp-112 of the β-hairpin. Δβh UvrB does not form a stable UvrB-DNA pre-incision complex and is inactive in UvrABC-mediated incision. However, Δβh UvrB is able to bind to UvrA and form a complex with UvrA and damaged DNA, competing with wild type UvrB. In addition, Δβh UvrB shows wild type-like ATPase activity in complex with UvrA that is stimulated by damaged DNA. In contrast to wild type UvrB, the ATPase activity of mutant UvrB does not lead to a destabilization of the damaged duplex. These results indicate that the conserved β-hairpin motif is a major factor in DNA binding. UvrB plays a major role in recognition and processing of DNA lesions during nucleotide excision repair. The crystal structure of UvrB revealed a similar fold as found in monomeric DNA helicases. Homology modeling suggested that the β-hairpin motif of UvrB might be involved in DNA binding (Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., and Kisker, C. (1999) EMBO J.18, 6899–6907). To determine a role of the β-hairpin ofBacillus caldotenax UvrB, we have constructed a deletion mutant, Δβh UvrB, which lacks residues Gln-97–Asp-112 of the β-hairpin. Δβh UvrB does not form a stable UvrB-DNA pre-incision complex and is inactive in UvrABC-mediated incision. However, Δβh UvrB is able to bind to UvrA and form a complex with UvrA and damaged DNA, competing with wild type UvrB. In addition, Δβh UvrB shows wild type-like ATPase activity in complex with UvrA that is stimulated by damaged DNA. In contrast to wild type UvrB, the ATPase activity of mutant UvrB does not lead to a destabilization of the damaged duplex. These results indicate that the conserved β-hairpin motif is a major factor in DNA binding. nucleotide excision repair base pair double-stranded DNA single-stranded DNA wild type Nucleotide excision repair (NER)1 is a highly conserved DNA repair pathway found in bacteria, yeast, and man (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995Google Scholar, 2Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Google Scholar). NER is remarkable because of the wide variety of chemically and structurally distinct DNA lesions that are substrates for this process (3Lloyd R.S. Van Houten B. Vos J.-M. DNA Damage Recognition. R. G. Landes Co., Biomedical Publishers, Austin, TX1995: 25-66Google Scholar). NER has been fully reconstituted from bacterial and mammalian proteins and can be viewed as four basic steps, damage recognition and processing, incision, repair synthesis, and ligation. One of the best-characterized NER systems is the UvrABC nuclease from Escherichia coli (4Van Houten B. Microbiol. Rev. 1990; 54: 18-51Google Scholar,5Grossman L. Lin C.I. Ahn Y. Nickoloff J.A. Hoekstra M.F. Nucleotide Excision Repair in Escherichia coli. 1. Humana Press Inc., Totowa, NJ1998: 11-27Google Scholar). Repair by UvrABC is initiated when the trimeric complex of UvrA2B recognizes the damaged site. It has been suggested that the UvrA2B complex may locate damage through a limited helicase activity (6Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3638-3642Google Scholar, 7Oh E.Y. Grossman L. J. Biol. Chem. 1989; 264: 1336-1343Google Scholar, 8Koo H.S. Claassen L. Grossman L. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1212-1216Google Scholar). However, more recent studies suggest that the strand-separating activity of the UvrA2B complex is not through a helicase-driven translocation step but in fact is due to local, relatively slow changes within the protein-DNA complex leading to a stable UvrB-DNA pre-incision complex and dissociation of the UvrA dimer (9Orren D.K. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5237-5241Google Scholar, 10Orren D.K. Sancar A. J. Biol. Chem. 1990; 265: 15796-15803Google Scholar, 11Gordienko I. Rupp W.D. EMBO J. 1997; 16: 889-895Google Scholar, 12Zou Y. Van Houten B. EMBO J. 1999; 18: 4889-4901Google Scholar). Once the UvrB-DNA complex has formed, UvrC, in what appears to be two different binding modes, first makes an incision four to five nucleotides 3′ to the modified nucleotide in an ATP-dependent step, which is followed by rapid incision seven nucleotides 5′ to the lesion in a step that does not require ATP hydrolysis (13Sancar A. Rupp W.D. Cell. 1983; 33: 249-260Google Scholar). Recent work from Goosen and co-workers (14Verhoeven E.E. van Kesteren M. Moolenaar G.F. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 5120-5123Google Scholar, 15Moolenaar G.F. Franken K.L.M.C. Dijkstra D.M. Thomas-Oates J.E. Visse R. van de Putte P. Goosen N. J. Biol. Chem. 1995; 270: 30508-30515Google Scholar, 16Moolenaar G.F. Franken K.L.M.C. van de Putte P. Goosen N. Mutat. Res. 1997; 385: 195-203Google Scholar, 17Moolenaar G.F. Herron M.F. Monaco V. van der Marel G.A. van Boom J.H. Visse R. Goosen N. J. Biol. Chem. 2000; 275: 8044-8050Google Scholar) strongly suggests, that in contrast to earlier reports (18Lin J.J. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6824-6828Google Scholar, 19Hsu D.S. Kim S.-T. Sun Q. Sancar A. J. Biol. Chem. 1995; 270: 8319-8327Google Scholar), UvrB has no intrinsic nuclease activity; 3′ incision is mediated by the N-terminal domain of UvrC and 5′ incision appears to be mediated by a nuclease center in the C-terminal domain of UvrC. After the incision reaction UvrD (helicase II) and DNA polymerase I are necessary and sufficient to release the excised oligonucleotide and allow UvrB and UvrC to participate in another round of incision (20Caron P.R. Kushner S.R. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4925-4929Google Scholar, 21Husain I. Van Houten B. Thomas D.C. Abdel-Monem M. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6774-6778Google Scholar).UvrB plays a central role in bacterial NER, participating in damage recognition, processing the DNA into a stable pre-incision complex, helping direct the activity of UvrC to perform the dual incisions, and finally staying bound to the non-damaged strand until being dislodged by DNA polymerase I (19Hsu D.S. Kim S.-T. Sun Q. Sancar A. J. Biol. Chem. 1995; 270: 8319-8327Google Scholar, 22Orren D.K. Selby C.P. Hearst J.E. Sancar A. J. Biol. Chem. 1992; 267: 780-788Google Scholar). UvrB contains six highly conserved sequence motifs, containing 10–40 amino acid residues each, that are found in all DNA helicases (23Moolenaar G.F. Visse R. Ortiz-Buysse M. Goosen N. van de Putte P. J. Mol. Biol. 1994; 240: 294-307Google Scholar, 24Goosen N. Moolenaar G.F. Visse R. van de Putte P. Eckstein F. Lilley D.M.J. Nucleic Acids and Molecular Biology. 12. Springer-Verlag, Berlin1998: 103-123Google Scholar). Three laboratories have independently solved the crystal structures of UvrB from thermophilic bacteria (25Theis K. Chen P.J. Skorvaga M. Van Houten B. Kisker C. EMBO J. 1999; 18: 6899-6907Google Scholar, 26Machius M. Henry L. Palnitkar M. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11717-11722Google Scholar, 27Nakagawa N. Sugahara M. Masui R. Kato R. Fukuyama K. Kuramitsu S. J. Biochem. (Tokyo). 1999; 126: 986-990Google Scholar, 28Theis K. Skorvaga M. Machius M. Nakagawa N. Van Houten B. Kisker C. Mutat. Res. 2000; 460: 277-300Google Scholar). UvrB is folded into five structural domains, 1a, 1b, 2, 3, and 4. The structure of domain 4, disordered in crystals of the full-length protein, has been determined separately (29Sohi M. Alexandrovich A. Moolenaar G. Visse R. Goosen N. Vernede X. Fontecilla-Camps J.C. Champness J. Sanderson M.R. FEBS Lett. 2000; 465: 161-164Google Scholar). Domains 1a and 3 contain the six helicase motifs, placing UvrB as a member of the helicase superfamily II (30Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. Nucleic Acids Res. 1989; 17: 4713-4730Google Scholar).Superpositioning of UvrB onto other helicase structures has revealed that UvrB domains 1a and 3 are structurally closely related to the monomeric helicase fold found in PcrA, NS3, and Rep (28Theis K. Skorvaga M. Machius M. Nakagawa N. Van Houten B. Kisker C. Mutat. Res. 2000; 460: 277-300Google Scholar). Domains 1b and 2 are unique to UvrB, the latter being a binding site for UvrA. Comparing the structure of UvrB with these helicase structures revealed that UvrB contains all the structural properties of a helicase that couple ATP binding and hydrolysis to domain motions. However, if UvrB binds DNA in a similar manner as observed in the DNA complexes of these helicases, then the translocated DNA strand would be partially covered by a flexible β-hairpin structure. This unique structural element (see Figs. 1, A and B) connecting domains 1a and 1b was found to be highly conserved in all bacterial species. The β-hairpin is held in place with respect to domain 1b by two salt bridges and hydrophobic interactions at the base and the tip of the hairpin. Similar β-hairpin motifs found in PcrA and RNA polymerase II are thought to be essential for the strand opening performed by these two proteins (31Soultanas P. Dillingham M.S. Velankar S.S. Wigley D.B. J. Mol. Biol. 1999; 290: 137-148Google Scholar, 32Cheetham G.M. Jeruzalmi D. Steitz T.A. Nature. 1999; 399: 80-83Google Scholar). We have previously shown that the DNA in the UvrB-DNA complex is partially melted over a 5-bp region (12Zou Y. Van Houten B. EMBO J. 1999; 18: 4889-4901Google Scholar). Based on these data, the UvrB structure, and comparisons to helicase structures and properties, a padlock binding mode for UvrB DNA interactions has been proposed in which UvrB wraps the β-hairpin around one DNA strand of partially unwound DNA in the pre-incision complex (25Theis K. Chen P.J. Skorvaga M. Van Houten B. Kisker C. EMBO J. 1999; 18: 6899-6907Google Scholar, 28Theis K. Skorvaga M. Machius M. Nakagawa N. Van Houten B. Kisker C. Mutat. Res. 2000; 460: 277-300Google Scholar). One critical test for this model is whether mutations in the β-hairpin affect binding and processing of damaged DNA.Only one known mutation has been made in this motif, Glu-99 in UvrB (of E. coli), which was found to decrease the incision activity of the UvrABC system. To investigate the role of the β-hairpin motif in more detail, we constructed a β-hairpin mutant that replaced residues 97–112 with a glycine residue. We report here that this β-hairpin deletion mutant is greatly reduced in its ability to support incision, bind to a damaged containing duplex, and destabilize a damage containing 26-mer but has retained the ability to hydrolyze ATP in a UvrA- and damaged DNA-dependent manner. Thus, ATP hydrolysis and formation of the UvrB-DNA pre-incision complex are uncoupled in this mutant. The ability of the mutant to form a UvrA2B-DNA complex and to hydrolyze ATP combined with its inability to form the UvrB-DNA pre-incision complex strongly suggests that the deleted residues are directly involved in DNA binding by UvrB.RESULTSTo test our padlock DNA binding model and the importance of the β-hairpin motif in the recognition of DNA damage, we have constructed a β-hairpin deletion mutant of the B. caldotenax UvrB protein, designed as Δβh UvrB, with amino acid residues from Gln97 to Asp112 removed and the resulting gap bridged by a glycine residue (Fig. 1, A and B). In the resulting deletion mutant only the upper half of the β-hairpin was removed. To test the properties of this mutant, we reconstituted the B. caldotenax UvrABC nuclease system with purified UvrA, UvrB, and UvrC (Fig. 1C), each obtained via intein fusion proteins.UvrABC-mediated Incision of a Fluorescein-containing 50-bp Duplex Using the UvrB β-Hairpin Deletion MutantWe first investigated the effect of Δβh UvrB on UvrABC endonuclease mediated-incision. The substrate was a 50-bp duplex containing a fluorescein moiety in the middle of the top strand (position F26, see Fig.2A), labeled at its 5′ terminus with [γ-32P]ATP. Results of the UvrABC endonuclease incision kinetics of F26-50 dsDNA are shown in Fig. 3. Panel A contains data for wild type UvrB, panel B contains data for Δβh UvrB, and panel C summarizes the incision kinetics. The results show that Δβh UvrB does not support UvrABC-mediated incision of substrate DNA. The residual incision of ≤5–6% represents the level of background for the substrate used. Clearly, deleting the β-hairpin of UvrB disrupts one of the steps that lead to incision of the damaged DNA.Figure 3Δβh UvrB does not support incision of a fluorescein containing a 50-bp duplex. The F26-50 dsDNA substrate (2 nm) (sequence shown in Fig. 2A with a 5′ terminally labeled modified strand) was incubated with UvrA (20 nm), UvrB (60 nm), and UvrC (50 nm) at 55 °C for 1 h. The samples were analyzed by PAGE under denaturating conditions. Panel A, wt UvrB; Panel B, Δβh UvrB. Panel C, kinetics of the incision reaction.View Large Image Figure ViewerDownload (PPT)Loading of the Δβh UvrB Protein onto the Site of DamageThe failure of Δβh UvrB to confer endonuclease activity to the UvrABC system might be due to failure to recognize the damage or failure to incise the damage after successful recognition. We used a gel mobility shift assay to test whether the intermediate between these processes, the UvrB-DNA pre-incision complex, is formed with the Δβh UvrB mutant (Figs. 4 and5). The Δβh UvrB protein does not form a stable complex with the damaged DNA neither at low concentrations (1–20 nm; Fig. 4A) nor at higher amounts (50–200 nm; Fig. 4B), whereas loading of wild type UvrB is very efficient, even at 5 nm (Fig.4A, lane 7). It is interesting to note that the band corresponding to the UvrA2-DNA complex (Fig.4B, lane 2) migrates slightly faster than the samples containing the Δβh UvrB protein (Fig. 4B,lanes 4–6). This slower mobility band probably represents the UvrA2Δβh UvrB-DNA complex. To further investigate whether Δβh UvrB is able to bind to UvrA, we have conducted competition experiments between the mutant and the wild type UvrB for binding to UvrA and F26-50 dsDNA. In these experiments (Fig. 5) there is a clear difference in mobility between the UvrA2-DNA and UvrA2Δβh UvrB-DNA complexes (Fig. 5, compare lane 2 with lanes 3–5). Increasing amounts of Δβh UvrB (10, 50, 100 nm) at a constant wild type UvrB concentration (5 nm) resulted in a significant reduction of the amount of wt UvrB-DNA complex (Fig.5, lanes 4–6 versus lane 8). This dominant negative effect of Δβh UvrB supports the idea that Δβh UvrB is properly folded and shows that it is capable of interacting with UvrA, resulting in the reduction of the amount of UvrA molecules available to interact with wild type UvrB.Figure 4Binding of Δβh UvrB to F26-50dsDNA. UvrA (20 nm) was incubated with various amounts of wild-type or mutant (Δβh) UvrB as indicated at 55 °C for 20 min in the presence of 2 nm F26-50 duplex DNA with the modified strand 5′ terminally labeled. The reaction mixtures were analyzed on 4% polyacrylamide native gels in the presence of ATP (1 mm) and MgCl2 (10 mm).Panel A, lower concentrations of Δβh UvrB (1–10 nm); panel B, higher concentrations of Δβh UvrB (50–200 nm).View Large Image Figure ViewerDownload (PPT)Figure 5Competition between wt and Δβh UvrB in binding to F26-50 dsDNA. UvrA (20 nm), wt UvrB (5 nm), and increasing amounts of Δβh UvrB (10–100 nm) were incubated at 55 °C for 20 min with 2 nm F26-50 dsDNA. The reaction mixtures were analyzed by 4% native PAGE using Tris-borate-EDTA running buffer with 1 mm ATP and 10 mmMgCl2.View Large Image Figure ViewerDownload (PPT)CD Spectra of Wild Type and the β-Hairpin Deletion Mutant UvrBFig. 6 shows CD spectra of wild type (filled ovals) and Δβh (open ovals) UvrB proteins, respectively. The results exhibit nearly identical CD spectra for both wild type and mutant proteins, indicating that the deletion of the β-hairpin motif in UvrB does not affect the global folding of the protein.Figure 6CD Spectra of wt and Δβh UvrB. CD spectra of wt UvrB (filled circles) and Δβh UvrB (open circles) collected in the range between 180 and 260 nm.View Large Image Figure ViewerDownload (PPT)Strand Destabilizing Activity of Δβh UvrBIn our padlock DNA binding model, we proposed that the β-hairpin of UvrB requires at least 5 base pairs of DNA to be disrupted so that the β-hairpin could be inserted between the strands of DNA. The limited strand opening by the UvrA2B complex has been shown previously to be important for dynamic recognition of DNA damage (11Gordienko I. Rupp W.D. EMBO J. 1997; 16: 889-895Google Scholar, 12Zou Y. Van Houten B. EMBO J. 1999; 18: 4889-4901Google Scholar) and has been called a limited helicase activity (6Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3638-3642Google Scholar, 7Oh E.Y. Grossman L. J. Biol. Chem. 1989; 264: 1336-1343Google Scholar). To evaluate the importance of the β-hairpin motif for the presumed helicase activity of the UvrA2B complex, we assayed Δβh UvrB in a strand destabilization assay that measures the release of a radioactively labeled 26-mer containing fluorescein annealed to a single-stranded DNA circle (M13mp19(+) strand). The results are shown in Fig.7, with kinetics of the 26-mer release summarized in panel C. Although wild type UvrB supports the release of the fluorescein-containing 26-mer very efficiently, reaching about 80% release of oligomer after 60 min, the β-hairpin deletion mutant has very low, if any, activity. It is critical to realize that the “release” of the oligomer is assayed after the addition of a stop buffer containing1% SDS and 0.1 m EDTA.Figure 7Δβh UvrB is not capable of destabilizing or incising a fluorescein-containing 26-mer annealed to ssHS1F-M13mp19(+) DNA. For the kinetics of release, HS1F-M13mp19(+) DNA (8 fmol) (sequence shown in Fig. 1B, with the modified strand 5′ terminally labeled) was incubated with UvrA (50 nm) and UvrB (100 nm), wt, or Δβh at 37 °C for the indicated periods of time. The reactions were terminated with stop buffer containing SDS, and the reaction mixtures were analyzed by 12% native PAGE. The figure shows the kinetics of 26-mer release by wt UvrB (panel A), Δβh UvrB (panel B), and graphic comparison of both wt and Δβh UvrB (panel C). Panel D, incision of a 26-mer containing fluorescein. HS1F-M13mp19(+) DNA (8 fmol) with a 5′ terminally labeled modified strand was incubated with UvrA (20 nm), UvrB (60 nm), and UvrC (50 nm) at 55 °C for 1 h. The samples were analyzed by PAGE under denaturing conditions.View Large Image Figure ViewerDownload (PPT)Incision of Strand-destabilizing Substrate by Δβh UvrBIf UvrB is capable of true strand displacement like a bona fidehelicase, then the displaced strand would be single-stranded. However, single-stranded damaged DNA is not a substrate for the UvrABC system. As can be clearly seen in Fig. 7D, the helix-destabilizing substrate, a 5′-labeled 26-mer containing a fluorescein adduct annealed to M13mp19 ssDNA, was incised by the UvrABC nuclease system. The incision efficiency supported by wild type UvrB was ∼55% (at 42 °C for 1 h; Fig. 7, panel D, lane 2), whereas the Δβh UvrB mutant did not support any incision of the 26-mer-fluorescein/M13 substrate. Based on this incision of the strand-displacement substrate with wild type UvrB (as part of the UvrABC endonuclease), we suggest that UvrA2B does not completely release the damage-containing 26-mer from a ssDNA circle until SDS is added as part of the stop buffer. Therefore, we feel it is inappropriate to call this activity a true helicase, and we suggest it is better to call this property of UvrA2UvrB a strand-destabilizing activity.ATPase Activity of Δβh UvrBIt has been shown previously that ATP binding/hydrolysis is absolutely required for NER (6Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3638-3642Google Scholar). In our padlock model (25Theis K. Chen P.J. Skorvaga M. Van Houten B. Kisker C. EMBO J. 1999; 18: 6899-6907Google Scholar) we suggest that the formation of a stable UvrB-DNA pre-incision complex requires free energy, which might be available either through ATP hydrolysis by UvrA2B or as a result of complex formation. To test whether the altered DNA binding properties of Δβh UvrB are due to an altered ATPase activity, we have examined this activity for both wild type UvrB and Δβh UvrB (TableI). By itself, Δβh UvrB has a very low ATPase activity at 37 °C (2.88 μmol of ATPase/min/mg of protein), similar to wild type UvrB (1.40 μmol/min/mg). In this respect, B. caldotenax UvrB resembles E. coliUvrB that has a cryptic ATPase activity. It has been shown that full ATPase activity of UvrB requires the presence of both UvrA and DNA (33Caron P.R. Grossman L. Nucleic Acids Res. 1988; 16: 10891-10902Google Scholar). Our data show that the ATPase activity of Δβh UvrB is not affected by deletion of the β-hairpin motif. In fact, in the presence of UV-irradiated DNA, the ATPase activity of the UvrA2ΔβhUvrB complex is higher than that of the UvrA2 wt UvrB complex (29, 22 μmol/min/mg, respectively). This is further evidence that UvrA and Δβh UvrB interact, as was suggested from our previous experiments (gel mobility shifts, CD spectra, helicase assay). The deletion of the β-hairpin does not interfere with the ATP hydrolysis by UvrB in the UvrA2B complex, but apparently the free energy of hydrolysis is not coupled to proper processing of the DNA that is necessary for UvrC binding and incision.Table IATPase activity of B. caldotenax UvrA, and UvrBSamplesATPase activityμmol of ATP hydrolyzed/μmol of protein/minUvrA13.2 ± 0.6UvrA + UV ↯>DNA17.0 ± 1.0UvrB1.4 ± 0.1UvrB + UV ↯>DNA1.4 ± 0.1UvrA + UvrB18.3 ± 1.1UvrA + UvrB + UV ↯>DNA22.0 ± 0.5Δβh UvrB2.8 ± 0.1UvrA + Δβh UvrB19.0 ± 0.6UvrA + Δβh UvrB + UV ↯>DNA29.0 ± 1.0 Open table in a new tab DISCUSSIONMutational analysis of UvrB has not yet identified which parts of UvrB are involved in DNA binding (reviewed in Ref. 28Theis K. Skorvaga M. Machius M. Nakagawa N. Van Houten B. Kisker C. Mutat. Res. 2000; 460: 277-300Google Scholar). One complication is that UvrB has an ATPase activity that is stimulated by DNA binding and is necessary for the formation of the pre-incision complex. Thus, defects of UvrB mutants defective in DNA binding might be due to defects in ATP binding/hydrolysis and vice versa. So far, mutants characterized for both properties showed either inactivation of both or no effect on either. Most of the mutations that affect DNA binding are located in the six highly conserved sequence motifs found in helicases of superfamily I and II. In a previous report (25Theis K. Chen P.J. Skorvaga M. Van Houten B. Kisker C. EMBO J. 1999; 18: 6899-6907Google Scholar) we presented a three-dimensional structure of the UvrB protein from the thermophilic bacterium B. caldotenax. The crystal structure of B. caldotenax UvrB has a significant level of similarity with that of helicase NS3 (34Kim J.L. Morgenstern K.A. Griffith J.P. Dwyer M.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure (Lond.). 1998; 6: 89-100Google Scholar). By superposition of B. caldotenax UvrB with the helicase domains of NS3 complexed with DNA, we have hypothesized a model for the UvrB-DNA pre-incision complex, which has a pivotal role in the mechanism of damage recognition by the UvrABC system. In our model we propose a padlock-like binding mode of UvrB to wrap around one DNA strand by inserting a β-hairpin between the two strands of DNA (28Theis K. Skorvaga M. Machius M. Nakagawa N. Van Houten B. Kisker C. Mutat. Res. 2000; 460: 277-300Google Scholar).To test our model and investigate the functional role of the β-hairpin motif, we constructed a β-hairpin deletion mutant (Δβh UvrB) in which residues 97–112 are replaced by a glycine, removing the tip of the β-hairpin. Data presented here show that the β-hairpin deletion mutant 1) is greatly reduced in its ability to support incision, 2) is unable to bind to a damage-containing duplex, 3) cannot destabilize a damage-containing 26-mer, and 4) has retained the ability to hydrolyze ATP in a UvrA and damaged DNA-dependent manner. Thus, functions of UvrB required for the formation of the UvrB-DNA complex, namely UvrA binding and ATP hydrolysis, are not disrupted in the deletion mutant. Nevertheless, Δβh UvrB is unable to form a stable complex with DNA, strongly suggesting that the deleted residues are directly involved in DNA binding.During the late 1980s and early 1990s Grossman and co-workers developed a “helicase scanning model” of damage recognition (6Oh E.Y. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3638-3642Google Scholar, 7Oh E.Y. Grossman L. J. Biol. Chem. 1989; 264: 1336-1343Google Scholar, 8Koo H.S. Claassen L. Grossman L. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1212-1216Google Scholar). In this model, UvrA2B can displace short oligonucleotides and also generate negative and positive supercoiled DNA as it migrates through the helix in search of damage. It was predicted that the helicase machine will stall at a lesion. However, UV was found to stimulate the negative/positive supercoiling, inconsistent with this earlier model (8Koo H.S. Claassen L. Grossman L. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1212-1216Google Scholar).Because of these discrepancies Gordienko and Rupp (11Gordienko I. Rupp W.D. EMBO J. 1997; 16: 889-895Google Scholar) developed a “damage-processing model” in which damage clearly increased the displacement of oligonucleotides (11Gordienko I. Rupp W.D. EMBO J. 1997; 16: 889-895Google Scholar). In this model UvrA2B finds damage by random diffusion. Once a lesion is encountered, the affinity of UvrA for DNA and UvrB is somehow weakened, and the dissociation of UvrA results in a UvrB-DNA complex. The DNA in this complex is greatly distorted, and it is believed that it is this step that requires ATP binding/hydrolysis to allow UvrB to facilitate cleavage upon UvrC binding. It is important to note that although these experiments have described the activity of UvrA2B as helicase-like, the DNA in these complexes is only destablized and not fully dissociated. In agreement with these results, we show here that the release of the 26-mer containing the damage does not occur until the addition of the loading buffer containing SDS. Because UvrC can still incise the destabilized strand, these results are inconsistent with a true helicase activity in which the strand is fully displaced from the complement strand. Our results in Fig. 7 demonstrate that deletion of the β-hairpin inhibits the helix destabilization step.The pre-incision complex between UvrB and damaged DNA is a key intermediate in excision repair linking damage recognition to the location of dual incision. Once the pre-incision complex is formed UvrB has to remain bound tightly to the DNA without translocating, ensuring precise incisions by UvrC and subsequent removal of the damaged fragment. In contrast to many non-sequence specific protein-DNA complexes, the UvrB-DNA pre-incision complex does not dissociate at high ionic strength, suggesting a hydrophobic mode of DNA binding. It has been suggested that UvrB might form favorable hydrophobic interactions with aromatic amino acid side chains and the DNA bases; however, this has never been directly determined (35Van Houten B. Snowden A. Bioessays. 1993; 15: 51-59Google Scholar) and awaits the solution of a co-crystal structure.We hypothesize that there are five critical regions in UvrB that are necessary and sufficient for DNA damage binding and processing: 1) a damage recognition pocket located at the base of the β-hairpin; 2) a flexible β-hairpin, which acts as a padlock to secure the non-damaged strand in place; 3) an ATP binding site, which facilitates conformational changes in UvrB; 4) the coiled-coiled C terminus of UvrB, which interacts with UvrC; and finally, 5) residues in domain 3 that contain helicase motifs IV-VI which help drive a conformational change in the DNA, leading to incision. How might the β-hairpin motif participate in allowing UvrB to bind and process damage? In our padlock DNA binding model of UvrB, the β-hairpin must first open to accept a strand of DNA and then close to lock one DNA strand; we favor the non-damaged strand, forming a stable UvrB-DNA interaction. Taking a closer look at the anatomy of the β-hairpin reveals four critical regions, three of which were removed in the Δβh mutant. The tip of the β-hairpin is hydrophobic in character and interacts with hydrophobic residues in domain Ib. Two salt bridges located in the middle of the hairpin provide further strength to the lock. Preserved in the Δβh mutant is an aromatic base containing several Tyr and Phe residues that are 100% conserved in all bacterial species examined to date. We propose that these residues are part of the damage recognition pocket. However, the interaction energy of these residues with the damaged strand without the strong padlock holding onto the non-damaged strand are apparently insufficient to provide sufficient binding energy in the Δβh mutant for producti" @default.
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• W2000675871 date "2002-01-01" @default.
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• W2000675871 title "The β-Hairpin Motif of UvrB Is Essential for DNA Binding, Damage Processing, and UvrC-mediated Incisions" @default.
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