- Email: [email protected]
Damage detection by the UvrABC pathway: Crystal structure of UvrB bound to fluorescein-adducted DNA
FEBS Letters 580 (2006) 6423–6427
Damage detection by the UvrABC pathway: Crystal structure of UvrB bound to fluorescein-adducted DNA Timothy R. Waters, Jitka Eryilmaz, Stella Geddes, Tracey E. Barrett* The School of Crystallography and the Institute for Structural Molecular Biology, Birkbeck College, Malet Street, London WC1E 7HX, United Kingdom Received 27 September 2006; revised 25 October 2006; accepted 25 October 2006 Available online 3 November 2006 Edited by Hans Eklund
Abstract UvrB is the damage recognition element of the highly conserved UvrABC pathway that functions in the removal of bulky DNA adducts. Pivotal to this is the formation of a damage detection complex that relies on the ability of UvrB to locate and sequester diverse lesions. Whilst structures of UvrB bound to DNA have recently been reported, none address the issue of lesion recognition. Here, we describe the crystal structure of UvrB bound to a pentanucleotide containing a single fluorescein-adducted thymine that reveals a unique mechanism for damage detection entirely dependent on the exclusion of lesions larger than an undamaged nucleotide. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: UvrABC; T-fluorescein; Protein crystallography
1. Introduction Nucleotide excision repair is a highly conserved pathway that efficiently corrects bulky DNA adducts that arise from exposure to both endogenous and exogenous agents. In bacteria, such lesions are targeted by the UvrABC mechanism comprising three proteins, UvrA, B and C, that act co-operatively to form tracking, damage detection and incision complexes [1– 4]. Formation of the tracking complex involves assembly of a UvrA2B2 heterotetramer that by means of a limited helicase activity driven by UvrB, is able to locate potential sites of damage typified by local distortions of the DNA duplex [5]. Once a lesion is detected, the UvrA–B complex undergoes a substantial conformational change in which the DNA becomes partially unwound in close proximity to the adduct and a tight UvrB–DNA complex formed, where the DNA becomes ‘‘wrapped’’ around UvrB [6]. The formation of this tight complex is thought to promote the dissociation of UvrA yielding a UvrB–DNA pre-incision complex although it is currently uncertain whether this intermediate involves a UvrB dimer or monomer. Assembly of the incision complex requires recruitment of UvrC, that is thought to displace one of the UvrB monomers, and is followed by dual incision of the damage containing DNA strand. UvrC initially cleaves the fourth or fifth phosphodiester bond 3 0 to the damage that is rapidly ensued by an incision at the eighth phosphodiester bond 5 0 * Corresponding author. Fax: +44 207 631 6803. E-mail address: [email protected] (T.E. Barrett).
to the lesion site. The resulting, highly stable UvrBC–DNA complex is dissociated by the DNA helicase UvrD, that also displaces the damage containing DNA strand. The resultant gapped duplex is then filled by DNA polymerase I and repair completed by DNA ligase. Despite extensive study of the UvrABC pathway, a number of key issues remain outstanding concerning the role and function of UvrB in the detection of damage and formation of incision complexes. Amongst these are whether, owing to an apparent lack of strand discrimination signals, the tracking process occurs in both strands. More importantly, it is still not known how lesions are recognised and subsequently presented to UvrC. Crystal structures of both apo UvrB and, more recently, DNA-bound forms have been published [7– 11] revealing the conformational changes in the UvrB molecule required for the formation of a DNA binding ‘‘competent’’ state and also the residues involved in forming protein–DNA contacts. Despite this, the key questions concerning lesion detection and recognition remain unanswered. In an attempt ˚ the crystal to address these, we have determined to 2.95 A structure of a ternary complex involving Bacillus subtilis UvrB, DNA containing a single fluorescein-adducted thymine (Tfluorescein) and ADP. This structure reveals that damage recognition is mediated by the front face of the b-hairpin and relies almost exclusively on the steric exclusion of lesions from the b-hairpin/domain 1b interface. Based on the position of the T-fluorescein adduct, we are able to explain how UvrB exhibits such a broad range of substrate specificity and speculate on how our structure may represent the precursor to the pre-incision complex.
2. Materials and methods The UvrB protein was purified as previously described [10]. The pentathymine oligonucleotide, (pTpTpXpTpT, where X is T-fluorescein that has a fluorescein group attached through a linker to carbon 5M of a thymine base, see Fig. 1A), was purchased from Eurogentec. Protein complexes were assembled as described for the UvrB–trithymine–ADP ternary complex [10] but with the trithymine substituted by the fluorescein-adducted pentathymine. Crystals grew from conditions containing 18–20% PEG 20 000, 0.1 M Tris–HCl (pH 8.0–9.0) and after cryo-cooling following brief incubation in crystallisation buffer to which 20% PEG 400 had been added, data were collected to ˚ at the ESRF (beamline ID23-2) (see Table 1 for details). Struc2.95 A ture determination was performed by molecular replacement (Molrep [12]) using the protein co-ordinates from our previous ternary complex obtained with trithymine (accession code 2D7D.pdb). Refinement was performed using Refmac5 with cycles of manual manipulation in O [13]. The current model consists of 5318 atoms with all geometrical
0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.10.051
6424
T.R. Waters et al. / FEBS Letters 580 (2006) 6423–6427
B
A
C
T1
T2 TF3 T4 PT5
Flexible linker
Fig. 1. (A) The molecular structure of T-fluorescein. (B) An overview of the UvrB molecule (represented as a molecular surface) showing the relative locations of the pentathymine molecule (cyan), the conserved b-hairpin (light green), domains 1a (yellow), 1b (grey), 2 (green) and 3 (pink). (C) A magnified view of the pentathymine molecule identifying the position of the T-fluorescein adducted nucleotide, TF3 (magenta), together with associated Fo Fc omit map density contoured at 2.5r. The location of the lesion reveals that the damage is extruded away from the UvrB molecule. PT5 denotes the 5 0 phosphate group of T5 that is the only visible moiety of this nucleotide. All figures were generated using Pymol (Delano Scientific, www.pymol.org).
Table 1 X-ray data collection and refinement statistics Data collection ˚) Cell dimensions (A Space group ˚) Resolution range (A Rmerge (%) Completeness I/SigI Multiplicity
a = 74.41 b = 95.60 c = 97.88 a = b = c = 90° P212121 68–2.95 9.4 (36.9)a 99.1 (99.3) 15.9 (5.2) 5.3
Structure refinement No of atoms (protein) 5185 No of atoms (DNA) 105 No of atoms (ADP) 27 28.32 Rfree (%) 20.53 Rcryst (%) P P P P Rmerge ¼ i I ij hI i ij = i hI j i, where j are the set of obserjj vations for each reflection i. P P Rcryst ¼ i kF o j jF c k= jF o j. Rfree = Rcryst for 5% of reflections omitted from refinement. a Numbers in parenthesis refer to the highest resolution shell.
parameters well within the ranges expected for structures at this resolution. The co-ordinates have been deposited with the protein databank (accession code 2NMV.pdb).
3. Results The pentathymine molecule (5 0 -pT1-T2-TF3-T4-T5) where TF3 is the fluorescein adducted thymine base could be clearly identified in preliminary maps. T1-T2-TF3 occupy positions at the entrance to the b-hairpin similar to those of trithymine in our previously published structure [10] consistent with the analogous nucleotides in the stem loop complex recently published by Truglio et al. [11] (Fig. 1B and 2). Density extending from carbon 5M of TF3 could also be observed up to the first peptide group of the largely aliphatic linker connecting the thymine base to the fluorescein moiety (Fig. 1C). In addition, density for the triple ring system of fluorescein could be detected at the base of the b-hairpin. T4 and T5 are located behind the b-hairpin where T5 is highly disordered beyond its 5 0
T.R. Waters et al. / FEBS Letters 580 (2006) 6423–6427
6425
3.1. The molecular basis for damage recognition Comparison of our complex with the UvrB–DNA stem loop structure of Truglio et al. that also contains a T-fluorescein adduct reveals a largely similar mode of binding with respect to protein–DNA contacts at the N-terminus of the b-hairpin involving residues Lys67, Ser91, Ser141 and Thr481 (Figs. 2 and 3A). However, the T-fluorescein in the stem loop structure is located away from the b-hairpin where it is intercalated in the duplex region of the DNA and unable to mediate interactions with UvrB (Fig. 2). Thus, the stem loop structure does not represent a complex in which the T-fluorescein is being recognised as damage, but as the authors state, reveals how UvrB binds to double stranded DNA with a 3 0 overhang. In our complex, the T-fluorescein nucleotide (TF3) is located at the entrance to the b-hairpin/domain 1b cavity where the base of the adjacent nucleotide (T4) packs against the N-terminus of the b-hairpin and is in contact with Tyr96 (Figs. 1C and 3A). The phosphate group of T5 (PT5) interacts with the side chain hydroxyls of both Tyr92 and Tyr93, but since it is the terminal nucleotide with a high degree of flexibility, the functional significance of these contacts is unclear. The thymine-fluorescein linker extending from carbon 5M (ordered to carbon 8) is extruded into the solvent region and although density for the remaining linker is absent, the triple ring system of the fluorescein moiety can be seen to occupy a solvent exposed recess created by residues Phe88, Asn116, Glu118 at the base of the b-hairpin and residues 67 to 74 in domain 1a (Fig. 3B). In addition to van der Waals contacts with the side
Fig. 2. Superposition of the pentathymine (light blue), trithymine (yellow) and stem–loop (grey) UvrB–DNA complexes. The fluorescein triple ring systems within the stem loop and pentathymine structures are shown in orange and magenta respectively.
phosphate group PT5 (Figs. 1C and 3A). Similar to our previous structure [10], density for ADP could be identified in the ATP binding site and for a helix–loop–helix dimer of a C-terminal proteolytic fragment of UvrB spanning domains 1a and 3. The interactions involving these moieties are similar to those previously described and will not be further commented upon.
A
TF3
TF4
TF3
Tyr96
TF4
PT5
PT5
Tyr93
Thr481
Lys67
Tyr92
Thr481
Ser91
Tyr93
Tyr92
Lys67 Ser91
Ser141
B
Tyr96
Ser141 Lys67
Lys67 Asn116 Phe88
Asn116 Phe88
Glu118
Glu118
Phe74 Ser75
Phe74 Ser75
Fig. 3. (A) Stereoview of the protein-DNA interactions involving the pentathymine molecule. Analysis of these contacts reveals a high degree of conservation in relation to the previous complexes including hydrogen bonds donated by Lys67, Ser91, Ser141 and Thr481. Within the b-hairpin, Tyr96, a key residue in damage recognition, stacks against TF4 of the pentathymine molecule where tyrosines 92 and 93 donate hydrogen bonds to the phosphate group of the terminal nucleotide T5. (B) Stereoview of the fluorescein triple ring system that is accommodated in a recess created by the helix spanning residues Lys67 to Ser75 in domain 1a and the b-hairpin base. The mode of recognition is largely non-specific where van der Waals contacts with Phe88 and Glu118 are observed in addition to two hydrogen bonds involving the exocyclic oxygens and side chain moieties of Asn116 and Ser75.
6426
T.R. Waters et al. / FEBS Letters 580 (2006) 6423–6427
chain moieties of Phe88 and Glu118, the exocyclic oxygens of the triple ring system make contacts with Asn116 and Ser75. Both the stem–loop structure and our complex confirm that a single DNA strand passes behind the b-hairpin. However, it would be impossible for a damaged nucleotide to be accommodated in the b-hairpin/domain 1b interface without substantial conformational changes in the UvrB molecule. We believe that our structure represents a UvrB damage detection complex and that large conformational changes do not occur when damage is encountered that would allow it to pass underneath the hairpin. We and others, have proposed that UvrB translocates along the DNA passing a single strand beneath the hairpin whilst moving in a 3 0 to 5 0 direction until it encounters an adduct [9,11]. As suggested by Truglio et al., [11] we believe that our structure is consistent with damage recognition occurring through steric exclusion of the lesion. The inability to pass behind the b-hairpin would thus cause translocation to arrest, hence signalling formation of the stable pre-incision complex, the following step in the reaction mechanism. Although it has been suggested that UvrA mediates pin opening for the formation of the UvrA–B-DNA complex as part of its loading mechanism and that this could in principle also be a factor in lesion recognition [9], it is difficult to envisage how remodelling of the region could produce a pocket or recess appropriately adapted for the task of recognising such a broad range of adducts. If, as we propose, the damage is simply extruded into the solvent following steric exclusion by the b-hairpin, there are potentially few limitations on adduct size or chemical composition. Furthermore, the role of the b-hairpin and comprising residues in damage recognition is well established [14,4]. In the mechanism proposed by Truglio et al., the damaged nucleotide would occupy a position equivalent to T4 in our complex that is directly 3 0 to the T-fluorescein nucleotide and whose base is in direct contact with Tyr96 of the b-hairpin (Fig. 3A). In our structure, it appears that without a substantial change in conformation, T-fluorescein could not be accommodated favourably in the T4 position owing to steric clashes between the triple ring system and hairpin base.
b-hairpin loop to sterically exclude lesions from the b-hairpin/domain 1b interface where the damage is extruded directly into the solvent region towards the hairpin base. Although Tyr96, a key residue in damage recognition [14,16,4], is in contact with T4 rather than TF3 in our structure, we suggest that steric clashes of the fluorescein group of TF3 and/or the flexible linker in certain conformations with the b-hairpin may make an analogous interaction unfavourable. The proximity of the damage to the b-hairpin, could thus explain the variations in the phosphodiester bond cleaved during the 3 0 incision where deviations of up to 3 phosphates have been reported [17]. Given that the association of the fluorescein group with the solvent exposed cavity at the hairpin base is largely non-specific, we would argue that these interactions are not fundamental to the exclusion process since conjugated aromatic ring systems are not components of all UvrABC substrates, but in cases where they are, may provide an additional level of recognition. Conversely, the b-hairpin and residues of which it is comprised, have been shown to have a key role in damage detection by several groups making our structure entirely consistent with not only the broad substrate specificity exhibited by the UvrABC pathway, but also its preference for bulky lesions such as benzo(a)-pyrene adducts, photoproducts and protein/peptide– DNA crosslinks [4,18]. These adducts would clearly be excluded from the b-hairpin/domain 1b interface. In keeping with this, small lesions such as abasic sites and mismatches are inefficiently repaired and whilst exclusion is unlikely to be an element in their recognition, may represent substrates that either result in pausing of the translocation process or give rise to duplexes that are more easily loaded onto UvrB due to local mispairing. The crystal structure of XPB, the archael/eukaryotic equivalent of UvrB, has recently been reported [19] and whilst structurally distinct still has to accommodate diverse lesions. It is possible that XPB uses a similar mechanism relying on the extrusion of damage into the solvent.
4. Discussion
References
Damage detection is one of the most important steps in any DNA repair mechanism and has been the subject of speculation in the UvrABC pathway due to the lack of appropriate structural data. Although the position of the damage has been suggested based on the previous structures and the available biochemical data [4,11], we report the first crystal structure of a UvrB–DNA damage containing complex that directly addresses the question of how chemically and structurally disparate lesions can be recognised. Furthermore, it is the first structural evidence that the b-hairpin is able to associate with both damaged and undamaged DNA. Our findings are supported by the recent studies of Malta et al. [15] who demonstrate that nucleotides directly 3 0 to the damage are buried within the UvrB molecule as judged by the fluorescence quenching of incorporated 2-amino purine. This is entirely consistent with the location of the damage in our complex where the adjacent nucleotides T4 and T5 are accommodated within the b-hairpin/domain 1b interface and thus have reduced solvent accessibility. Our complex further reveals that the key to diverse substrate specificity lies in the ability of the conserved
Acknowledgements: We thank Dr. Ajit Basak for help with data collection. This work was funded by a BBSRC Project Grant to T.E.B.
[1] Van Houten, B. (1990) Nucleotide excision repair in Escherichia coli. Microbiol. Rev. 54, 18–51. [2] Sancar, A. (1996) DNA excision repair. Annu. Rev. Biochem. 65, 43–81. [3] Goosen, N., Moolenaar, G.F., Visse, R. and van de Putte, P. (1998) in: Nucleic Acids and Molecular Biology: DNA Repair (Eckstein, F. and Lilley, D.M.G., Eds.), pp. 101–123, Springer Verlag, Berlin. [4] Van Houten, B., Croteau, D.L., Dellavecchia, M.J., Wang, H. and Kisker, C. (2005) ‘close-fitting sleeves’: DNA damage recognition by the uvrabc nuclease system. Mutat. Res. 577, 92– 117. [5] Verhoeven, E.E., Wyman, C., Moolenaar, G.F. and Goosen, N. (2002) The presence of two uvrb subunits in the uvrab complex ensures damage detection in both DNA strands. EMBO J. 21, 4196–4205. [6] Verhoeven, E.E., Wyman, C., Moolenaar, G.F., Hoeijmakers, J.H. and Goosen, N. (2001) Architecture of nucleotide excision repair complexes: DNA is wrapped by uvrb before and after damage recognition. EMBO J. 20, 601–611. [7] Machius, M., Henry, L., Palnitkar, M. and Deisenhofer, J. (1999) Crystal structure of the DNA nucleotide excision repair enzyme uvrb from Thermus thermophilus. Proc. Natl. Acad. Sci. USA 96, 11717–11722.
T.R. Waters et al. / FEBS Letters 580 (2006) 6423–6427 [8] Nakagawa, N., Sugahara, M., Masui, R., Kato, R., Fukuyama, K. and Kuramitsu, S. (1999) Crystal structure of Thermus thermophilus hb8 uvrb protein, a key enzyme of nucleotide excision repair. J. Biochem. (Tokyo) 126, 986–990. [9] Theis, K., Chen, P.J., Skorvaga, M., Van Houten, B. and Kisker, C. (1999) Crystal structure of uvrb, a DNA helicase adapted for nucleotide excision repair. EMBO J. 18, 6899–6907. [10] Eryilmaz, J., Ceschini, S., Ryan, J., Geddes, S., Waters, T.R. and Barrett, T.E. (2006) Structural insights into the cryptic ATP-ase activity of uvrb. J. Mol. Biol. 357, 62–72. [11] Truglio, J.J., Karakas, E., Rhau, B., Wang, H., Dellavecchia, M.J., Van Houten, B. and Kisker, C. (2006) Structural basis for DNA recognition and processing by uvrb. Nat. Struct. Biol. 13, 360–364. [12] Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. (1994) Acta Crystallogr. D50, 760–763. [13] Jones, T.A., Zou, J.Y., Cowan, S.W. and Kjeldgaard, M. (1991) Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119. [14] Moolenaar, G.F., Hoglund, L. and Goosen, N. (2001) Clue to damage recognition by uvrb: residues in the beta-hairpin struc-
6427
[15] [16]
[17]
[18]
[19]
ture prevent binding to non-damaged DNA. EMBO J. 20, 6140– 6149. Malta, E., Moolenaar, G.F. and Goosen, N. (2006) Base flipping in nucleotide excision repair. J. Biol. Chem. 281, 2184–2194. Skorvaga, M., DellaVecchia, M.J., Croteau, D.L., Theis, K., Truglio, J.J., Mandavilli, B.S., Kisker, C. and Van Houten, B. (2004) Identification of residues within uvrb that are important for efficient DNA binding and damage processing. J. Biol. Chem. 279, 51574–51580. Hoare, S., Zou, Y., Purohit, R., Krishnasamy, M., Skorvaga, M., Van Houten, B., Geacintov, N.E. and Basu, A.K. (2000) Differential incision of bulky carcinogenic-DNA adducts by the uvrabc nuclease: Comparison of incision rates and the interactions of uvr subunits with lesions of different structures. Biochemistry 39, 12252–12261. Minko, I.G., Kurtz, A.J., Croteau, D.L., Van Houten, B., Harris, T.M. and Lloyd, R.S. (2005) Initiation of repair of DNA– polypeptide cross-links by the uvrabc nuclease. Biochemistry 44, 3000–3009. Fan, L., Arvai, A.S., Cooper, P.K., Iwai, S., Hanaoka, F. and Tainer, J.A. (2006) Conserved xpb core structure and motifs for DNA unwinding: implications for pathway selection of transcription or excision repair. Mol. Cell 22, 27–37.
Damage detection by the UvrABC pathway: Crystal structure of UvrB bound to fluorescein-adducted DNA Timothy R. Waters, Jitka Eryilmaz, Stella Geddes, Tracey E. Barrett* The School of Crystallography and the Institute for Structural Molecular Biology, Birkbeck College, Malet Street, London WC1E 7HX, United Kingdom Received 27 September 2006; revised 25 October 2006; accepted 25 October 2006 Available online 3 November 2006 Edited by Hans Eklund
Abstract UvrB is the damage recognition element of the highly conserved UvrABC pathway that functions in the removal of bulky DNA adducts. Pivotal to this is the formation of a damage detection complex that relies on the ability of UvrB to locate and sequester diverse lesions. Whilst structures of UvrB bound to DNA have recently been reported, none address the issue of lesion recognition. Here, we describe the crystal structure of UvrB bound to a pentanucleotide containing a single fluorescein-adducted thymine that reveals a unique mechanism for damage detection entirely dependent on the exclusion of lesions larger than an undamaged nucleotide. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: UvrABC; T-fluorescein; Protein crystallography
1. Introduction Nucleotide excision repair is a highly conserved pathway that efficiently corrects bulky DNA adducts that arise from exposure to both endogenous and exogenous agents. In bacteria, such lesions are targeted by the UvrABC mechanism comprising three proteins, UvrA, B and C, that act co-operatively to form tracking, damage detection and incision complexes [1– 4]. Formation of the tracking complex involves assembly of a UvrA2B2 heterotetramer that by means of a limited helicase activity driven by UvrB, is able to locate potential sites of damage typified by local distortions of the DNA duplex [5]. Once a lesion is detected, the UvrA–B complex undergoes a substantial conformational change in which the DNA becomes partially unwound in close proximity to the adduct and a tight UvrB–DNA complex formed, where the DNA becomes ‘‘wrapped’’ around UvrB [6]. The formation of this tight complex is thought to promote the dissociation of UvrA yielding a UvrB–DNA pre-incision complex although it is currently uncertain whether this intermediate involves a UvrB dimer or monomer. Assembly of the incision complex requires recruitment of UvrC, that is thought to displace one of the UvrB monomers, and is followed by dual incision of the damage containing DNA strand. UvrC initially cleaves the fourth or fifth phosphodiester bond 3 0 to the damage that is rapidly ensued by an incision at the eighth phosphodiester bond 5 0 * Corresponding author. Fax: +44 207 631 6803. E-mail address: [email protected] (T.E. Barrett).
to the lesion site. The resulting, highly stable UvrBC–DNA complex is dissociated by the DNA helicase UvrD, that also displaces the damage containing DNA strand. The resultant gapped duplex is then filled by DNA polymerase I and repair completed by DNA ligase. Despite extensive study of the UvrABC pathway, a number of key issues remain outstanding concerning the role and function of UvrB in the detection of damage and formation of incision complexes. Amongst these are whether, owing to an apparent lack of strand discrimination signals, the tracking process occurs in both strands. More importantly, it is still not known how lesions are recognised and subsequently presented to UvrC. Crystal structures of both apo UvrB and, more recently, DNA-bound forms have been published [7– 11] revealing the conformational changes in the UvrB molecule required for the formation of a DNA binding ‘‘competent’’ state and also the residues involved in forming protein–DNA contacts. Despite this, the key questions concerning lesion detection and recognition remain unanswered. In an attempt ˚ the crystal to address these, we have determined to 2.95 A structure of a ternary complex involving Bacillus subtilis UvrB, DNA containing a single fluorescein-adducted thymine (Tfluorescein) and ADP. This structure reveals that damage recognition is mediated by the front face of the b-hairpin and relies almost exclusively on the steric exclusion of lesions from the b-hairpin/domain 1b interface. Based on the position of the T-fluorescein adduct, we are able to explain how UvrB exhibits such a broad range of substrate specificity and speculate on how our structure may represent the precursor to the pre-incision complex.
2. Materials and methods The UvrB protein was purified as previously described [10]. The pentathymine oligonucleotide, (pTpTpXpTpT, where X is T-fluorescein that has a fluorescein group attached through a linker to carbon 5M of a thymine base, see Fig. 1A), was purchased from Eurogentec. Protein complexes were assembled as described for the UvrB–trithymine–ADP ternary complex [10] but with the trithymine substituted by the fluorescein-adducted pentathymine. Crystals grew from conditions containing 18–20% PEG 20 000, 0.1 M Tris–HCl (pH 8.0–9.0) and after cryo-cooling following brief incubation in crystallisation buffer to which 20% PEG 400 had been added, data were collected to ˚ at the ESRF (beamline ID23-2) (see Table 1 for details). Struc2.95 A ture determination was performed by molecular replacement (Molrep [12]) using the protein co-ordinates from our previous ternary complex obtained with trithymine (accession code 2D7D.pdb). Refinement was performed using Refmac5 with cycles of manual manipulation in O [13]. The current model consists of 5318 atoms with all geometrical
0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.10.051
6424
T.R. Waters et al. / FEBS Letters 580 (2006) 6423–6427
B
A
C
T1
T2 TF3 T4 PT5
Flexible linker
Fig. 1. (A) The molecular structure of T-fluorescein. (B) An overview of the UvrB molecule (represented as a molecular surface) showing the relative locations of the pentathymine molecule (cyan), the conserved b-hairpin (light green), domains 1a (yellow), 1b (grey), 2 (green) and 3 (pink). (C) A magnified view of the pentathymine molecule identifying the position of the T-fluorescein adducted nucleotide, TF3 (magenta), together with associated Fo Fc omit map density contoured at 2.5r. The location of the lesion reveals that the damage is extruded away from the UvrB molecule. PT5 denotes the 5 0 phosphate group of T5 that is the only visible moiety of this nucleotide. All figures were generated using Pymol (Delano Scientific, www.pymol.org).
Table 1 X-ray data collection and refinement statistics Data collection ˚) Cell dimensions (A Space group ˚) Resolution range (A Rmerge (%) Completeness I/SigI Multiplicity
a = 74.41 b = 95.60 c = 97.88 a = b = c = 90° P212121 68–2.95 9.4 (36.9)a 99.1 (99.3) 15.9 (5.2) 5.3
Structure refinement No of atoms (protein) 5185 No of atoms (DNA) 105 No of atoms (ADP) 27 28.32 Rfree (%) 20.53 Rcryst (%) P P P P Rmerge ¼ i I ij hI i ij = i hI j i, where j are the set of obserjj vations for each reflection i. P P Rcryst ¼ i kF o j jF c k= jF o j. Rfree = Rcryst for 5% of reflections omitted from refinement. a Numbers in parenthesis refer to the highest resolution shell.
parameters well within the ranges expected for structures at this resolution. The co-ordinates have been deposited with the protein databank (accession code 2NMV.pdb).
3. Results The pentathymine molecule (5 0 -pT1-T2-TF3-T4-T5) where TF3 is the fluorescein adducted thymine base could be clearly identified in preliminary maps. T1-T2-TF3 occupy positions at the entrance to the b-hairpin similar to those of trithymine in our previously published structure [10] consistent with the analogous nucleotides in the stem loop complex recently published by Truglio et al. [11] (Fig. 1B and 2). Density extending from carbon 5M of TF3 could also be observed up to the first peptide group of the largely aliphatic linker connecting the thymine base to the fluorescein moiety (Fig. 1C). In addition, density for the triple ring system of fluorescein could be detected at the base of the b-hairpin. T4 and T5 are located behind the b-hairpin where T5 is highly disordered beyond its 5 0
T.R. Waters et al. / FEBS Letters 580 (2006) 6423–6427
6425
3.1. The molecular basis for damage recognition Comparison of our complex with the UvrB–DNA stem loop structure of Truglio et al. that also contains a T-fluorescein adduct reveals a largely similar mode of binding with respect to protein–DNA contacts at the N-terminus of the b-hairpin involving residues Lys67, Ser91, Ser141 and Thr481 (Figs. 2 and 3A). However, the T-fluorescein in the stem loop structure is located away from the b-hairpin where it is intercalated in the duplex region of the DNA and unable to mediate interactions with UvrB (Fig. 2). Thus, the stem loop structure does not represent a complex in which the T-fluorescein is being recognised as damage, but as the authors state, reveals how UvrB binds to double stranded DNA with a 3 0 overhang. In our complex, the T-fluorescein nucleotide (TF3) is located at the entrance to the b-hairpin/domain 1b cavity where the base of the adjacent nucleotide (T4) packs against the N-terminus of the b-hairpin and is in contact with Tyr96 (Figs. 1C and 3A). The phosphate group of T5 (PT5) interacts with the side chain hydroxyls of both Tyr92 and Tyr93, but since it is the terminal nucleotide with a high degree of flexibility, the functional significance of these contacts is unclear. The thymine-fluorescein linker extending from carbon 5M (ordered to carbon 8) is extruded into the solvent region and although density for the remaining linker is absent, the triple ring system of the fluorescein moiety can be seen to occupy a solvent exposed recess created by residues Phe88, Asn116, Glu118 at the base of the b-hairpin and residues 67 to 74 in domain 1a (Fig. 3B). In addition to van der Waals contacts with the side
Fig. 2. Superposition of the pentathymine (light blue), trithymine (yellow) and stem–loop (grey) UvrB–DNA complexes. The fluorescein triple ring systems within the stem loop and pentathymine structures are shown in orange and magenta respectively.
phosphate group PT5 (Figs. 1C and 3A). Similar to our previous structure [10], density for ADP could be identified in the ATP binding site and for a helix–loop–helix dimer of a C-terminal proteolytic fragment of UvrB spanning domains 1a and 3. The interactions involving these moieties are similar to those previously described and will not be further commented upon.
A
TF3
TF4
TF3
Tyr96
TF4
PT5
PT5
Tyr93
Thr481
Lys67
Tyr92
Thr481
Ser91
Tyr93
Tyr92
Lys67 Ser91
Ser141
B
Tyr96
Ser141 Lys67
Lys67 Asn116 Phe88
Asn116 Phe88
Glu118
Glu118
Phe74 Ser75
Phe74 Ser75
Fig. 3. (A) Stereoview of the protein-DNA interactions involving the pentathymine molecule. Analysis of these contacts reveals a high degree of conservation in relation to the previous complexes including hydrogen bonds donated by Lys67, Ser91, Ser141 and Thr481. Within the b-hairpin, Tyr96, a key residue in damage recognition, stacks against TF4 of the pentathymine molecule where tyrosines 92 and 93 donate hydrogen bonds to the phosphate group of the terminal nucleotide T5. (B) Stereoview of the fluorescein triple ring system that is accommodated in a recess created by the helix spanning residues Lys67 to Ser75 in domain 1a and the b-hairpin base. The mode of recognition is largely non-specific where van der Waals contacts with Phe88 and Glu118 are observed in addition to two hydrogen bonds involving the exocyclic oxygens and side chain moieties of Asn116 and Ser75.
6426
T.R. Waters et al. / FEBS Letters 580 (2006) 6423–6427
chain moieties of Phe88 and Glu118, the exocyclic oxygens of the triple ring system make contacts with Asn116 and Ser75. Both the stem–loop structure and our complex confirm that a single DNA strand passes behind the b-hairpin. However, it would be impossible for a damaged nucleotide to be accommodated in the b-hairpin/domain 1b interface without substantial conformational changes in the UvrB molecule. We believe that our structure represents a UvrB damage detection complex and that large conformational changes do not occur when damage is encountered that would allow it to pass underneath the hairpin. We and others, have proposed that UvrB translocates along the DNA passing a single strand beneath the hairpin whilst moving in a 3 0 to 5 0 direction until it encounters an adduct [9,11]. As suggested by Truglio et al., [11] we believe that our structure is consistent with damage recognition occurring through steric exclusion of the lesion. The inability to pass behind the b-hairpin would thus cause translocation to arrest, hence signalling formation of the stable pre-incision complex, the following step in the reaction mechanism. Although it has been suggested that UvrA mediates pin opening for the formation of the UvrA–B-DNA complex as part of its loading mechanism and that this could in principle also be a factor in lesion recognition [9], it is difficult to envisage how remodelling of the region could produce a pocket or recess appropriately adapted for the task of recognising such a broad range of adducts. If, as we propose, the damage is simply extruded into the solvent following steric exclusion by the b-hairpin, there are potentially few limitations on adduct size or chemical composition. Furthermore, the role of the b-hairpin and comprising residues in damage recognition is well established [14,4]. In the mechanism proposed by Truglio et al., the damaged nucleotide would occupy a position equivalent to T4 in our complex that is directly 3 0 to the T-fluorescein nucleotide and whose base is in direct contact with Tyr96 of the b-hairpin (Fig. 3A). In our structure, it appears that without a substantial change in conformation, T-fluorescein could not be accommodated favourably in the T4 position owing to steric clashes between the triple ring system and hairpin base.
b-hairpin loop to sterically exclude lesions from the b-hairpin/domain 1b interface where the damage is extruded directly into the solvent region towards the hairpin base. Although Tyr96, a key residue in damage recognition [14,16,4], is in contact with T4 rather than TF3 in our structure, we suggest that steric clashes of the fluorescein group of TF3 and/or the flexible linker in certain conformations with the b-hairpin may make an analogous interaction unfavourable. The proximity of the damage to the b-hairpin, could thus explain the variations in the phosphodiester bond cleaved during the 3 0 incision where deviations of up to 3 phosphates have been reported [17]. Given that the association of the fluorescein group with the solvent exposed cavity at the hairpin base is largely non-specific, we would argue that these interactions are not fundamental to the exclusion process since conjugated aromatic ring systems are not components of all UvrABC substrates, but in cases where they are, may provide an additional level of recognition. Conversely, the b-hairpin and residues of which it is comprised, have been shown to have a key role in damage detection by several groups making our structure entirely consistent with not only the broad substrate specificity exhibited by the UvrABC pathway, but also its preference for bulky lesions such as benzo(a)-pyrene adducts, photoproducts and protein/peptide– DNA crosslinks [4,18]. These adducts would clearly be excluded from the b-hairpin/domain 1b interface. In keeping with this, small lesions such as abasic sites and mismatches are inefficiently repaired and whilst exclusion is unlikely to be an element in their recognition, may represent substrates that either result in pausing of the translocation process or give rise to duplexes that are more easily loaded onto UvrB due to local mispairing. The crystal structure of XPB, the archael/eukaryotic equivalent of UvrB, has recently been reported [19] and whilst structurally distinct still has to accommodate diverse lesions. It is possible that XPB uses a similar mechanism relying on the extrusion of damage into the solvent.
4. Discussion
References
Damage detection is one of the most important steps in any DNA repair mechanism and has been the subject of speculation in the UvrABC pathway due to the lack of appropriate structural data. Although the position of the damage has been suggested based on the previous structures and the available biochemical data [4,11], we report the first crystal structure of a UvrB–DNA damage containing complex that directly addresses the question of how chemically and structurally disparate lesions can be recognised. Furthermore, it is the first structural evidence that the b-hairpin is able to associate with both damaged and undamaged DNA. Our findings are supported by the recent studies of Malta et al. [15] who demonstrate that nucleotides directly 3 0 to the damage are buried within the UvrB molecule as judged by the fluorescence quenching of incorporated 2-amino purine. This is entirely consistent with the location of the damage in our complex where the adjacent nucleotides T4 and T5 are accommodated within the b-hairpin/domain 1b interface and thus have reduced solvent accessibility. Our complex further reveals that the key to diverse substrate specificity lies in the ability of the conserved
Acknowledgements: We thank Dr. Ajit Basak for help with data collection. This work was funded by a BBSRC Project Grant to T.E.B.
[1] Van Houten, B. (1990) Nucleotide excision repair in Escherichia coli. Microbiol. Rev. 54, 18–51. [2] Sancar, A. (1996) DNA excision repair. Annu. Rev. Biochem. 65, 43–81. [3] Goosen, N., Moolenaar, G.F., Visse, R. and van de Putte, P. (1998) in: Nucleic Acids and Molecular Biology: DNA Repair (Eckstein, F. and Lilley, D.M.G., Eds.), pp. 101–123, Springer Verlag, Berlin. [4] Van Houten, B., Croteau, D.L., Dellavecchia, M.J., Wang, H. and Kisker, C. (2005) ‘close-fitting sleeves’: DNA damage recognition by the uvrabc nuclease system. Mutat. Res. 577, 92– 117. [5] Verhoeven, E.E., Wyman, C., Moolenaar, G.F. and Goosen, N. (2002) The presence of two uvrb subunits in the uvrab complex ensures damage detection in both DNA strands. EMBO J. 21, 4196–4205. [6] Verhoeven, E.E., Wyman, C., Moolenaar, G.F., Hoeijmakers, J.H. and Goosen, N. (2001) Architecture of nucleotide excision repair complexes: DNA is wrapped by uvrb before and after damage recognition. EMBO J. 20, 601–611. [7] Machius, M., Henry, L., Palnitkar, M. and Deisenhofer, J. (1999) Crystal structure of the DNA nucleotide excision repair enzyme uvrb from Thermus thermophilus. Proc. Natl. Acad. Sci. USA 96, 11717–11722.
T.R. Waters et al. / FEBS Letters 580 (2006) 6423–6427 [8] Nakagawa, N., Sugahara, M., Masui, R., Kato, R., Fukuyama, K. and Kuramitsu, S. (1999) Crystal structure of Thermus thermophilus hb8 uvrb protein, a key enzyme of nucleotide excision repair. J. Biochem. (Tokyo) 126, 986–990. [9] Theis, K., Chen, P.J., Skorvaga, M., Van Houten, B. and Kisker, C. (1999) Crystal structure of uvrb, a DNA helicase adapted for nucleotide excision repair. EMBO J. 18, 6899–6907. [10] Eryilmaz, J., Ceschini, S., Ryan, J., Geddes, S., Waters, T.R. and Barrett, T.E. (2006) Structural insights into the cryptic ATP-ase activity of uvrb. J. Mol. Biol. 357, 62–72. [11] Truglio, J.J., Karakas, E., Rhau, B., Wang, H., Dellavecchia, M.J., Van Houten, B. and Kisker, C. (2006) Structural basis for DNA recognition and processing by uvrb. Nat. Struct. Biol. 13, 360–364. [12] Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. (1994) Acta Crystallogr. D50, 760–763. [13] Jones, T.A., Zou, J.Y., Cowan, S.W. and Kjeldgaard, M. (1991) Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119. [14] Moolenaar, G.F., Hoglund, L. and Goosen, N. (2001) Clue to damage recognition by uvrb: residues in the beta-hairpin struc-
6427
[15] [16]
[17]
[18]
[19]
ture prevent binding to non-damaged DNA. EMBO J. 20, 6140– 6149. Malta, E., Moolenaar, G.F. and Goosen, N. (2006) Base flipping in nucleotide excision repair. J. Biol. Chem. 281, 2184–2194. Skorvaga, M., DellaVecchia, M.J., Croteau, D.L., Theis, K., Truglio, J.J., Mandavilli, B.S., Kisker, C. and Van Houten, B. (2004) Identification of residues within uvrb that are important for efficient DNA binding and damage processing. J. Biol. Chem. 279, 51574–51580. Hoare, S., Zou, Y., Purohit, R., Krishnasamy, M., Skorvaga, M., Van Houten, B., Geacintov, N.E. and Basu, A.K. (2000) Differential incision of bulky carcinogenic-DNA adducts by the uvrabc nuclease: Comparison of incision rates and the interactions of uvr subunits with lesions of different structures. Biochemistry 39, 12252–12261. Minko, I.G., Kurtz, A.J., Croteau, D.L., Van Houten, B., Harris, T.M. and Lloyd, R.S. (2005) Initiation of repair of DNA– polypeptide cross-links by the uvrabc nuclease. Biochemistry 44, 3000–3009. Fan, L., Arvai, A.S., Cooper, P.K., Iwai, S., Hanaoka, F. and Tainer, J.A. (2006) Conserved xpb core structure and motifs for DNA unwinding: implications for pathway selection of transcription or excision repair. Mol. Cell 22, 27–37.