Tài liệu Báo cáo Y học: Dynamic mechanism of nick recognition by DNA ligase ppt

REVIEW ARTICLE Dynamic mechanism of nick recognition by DNA ligase Alexei V. Cherepanov* and Simon de Vries Kluyver Department of Biotechnology, Delft University of Technology, Delft, the Netherlands DNA ligases are the enzymes responsible for the repair of single-stranded and double-stranded nicks in dsDNA. DNA ligases are structurally similar, possibly sharing a common molecular mechanism of nick recognition and ligation catalysis. This mechanism remains unclear, in part because the structure of ligase in complex with dsDNA has yet to be solved. DNA ligases share common structural elements with DNA polymerases, which have been cocrystallized with dsDNA. Based on the observed DNA polymerase–dsDNA interactions, we propose a mechanism for recognition of a single-stranded nick by DNA ligase. According to this mechanism, ligase induces a B-to-A DNA helix transition of the enzyme-bound dsDNA motif, which results in DNA contraction, bending and unwinding. For non-nicked dsDNA, this transition is reversible, leading to dissociation of the enzyme. For a nicked dsDNA substrate, the con- traction of the enzyme-bound DNA motif (a) triggers an opened–closed conformational change of the enzyme, and (b) forces the motif to accommodate the strained A/B-form hybrid conformation, in which the nicked strand tends to retain a B-type helix, while the non-nicked strand tends to form a shortened A-type helix. We propose that this con- formation is the catalytically competent transition state, which leads to the formation of the DNA–AMP interme- diate and to the subsequent sealing of the nick. Keywords: DNA ligase; nick recognition; A-form DNA; A/ B-form DNA hybrid; protein–DNA interactions; B-A DNA helix transition. DNA ligases are the enzymes that catalyze the joining of single- and double-stranded nicks in dsDNA [1]. These enzymes play a pivotal role in replication, sealing the nicks in the lagging DNA strand [2–5]. They also participate in DNA excision [6–8], double-strand break repair [9–12] and take part in DNA recombination [10,13–15]. The mechan- ism of enzyme catalysis (Scheme 1) includes three main steps: (1) covalent binding of the nucleoside monophos- phate, AMP or GMP, via the e-amino lysyl phosphorami- date bond, (2) transfer of the nucleotidyl moiety onto the 5¢-phosphate end of the nick, forming an inverted pyro- phosphate bridging structure, A(G)ppN and (3) formation of the phosphodiester bond between the 3¢-OH and the 5¢-phosphate ends of the nick, releasing the nucleotide. Scheme 1. Mechanism of the ATP-dependent end-joining activity of T4 DNA ligase. nds- DNA, dsDNA containing a 5¢-phosphorylated nick. n-MgAMP-dsDNA, nicked dsDNA adenylylated at the 5¢-phosphate of the nick. Correspondence to S. de Vries, Kluyver Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, the Netherlands Tel.: + 31 15 2785139, Fax: + 31 15 2782355, E-mail: S.deVries@tnw.tudelft.nl Abbreviations: EMSA, electrophoretic mobility shift assay. Enzymes: DNA ligase (EC 6.5.1.1). *Present address: Metalloprotein & Protein Engineering Group, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, PO Box 9502, 2300 RA Leiden, the Netherlands. (Received 8 July 2002, accepted 11 October 2002) Eur. J. Biochem. 269, 5993–5999 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03309.x STRUCTURE OF DNA LIGASES The crystal structures of several ATP- and NAD + -depend- ent DNA ligases have been solved: the bacteriophage T7 DNA ligase complex with ATP [17,18], the enzyme–AMP covalent complexes of the eukaryotic DNA ligase from Chlorella virus [19] and of the thermophilic bacterium Thermus filiformis [20,21]. In addition, the structure of the adenylylation domain of the NAD + -dependent DNA ligase from Bacillus stearothermophilus has been determined [22]. Analyses indicate that these proteins are very similar [23,24], and that the minimal catalytic core of the ATP-dependent DNA ligase consists of two structurally conserved domains (Fig. 1). The N-terminal domain 1 (blue and green regions, Fig. 1) contains the active site, where the adenylylation of the enzyme takes place. Within domain 1, a smaller subdomain (1c) can be distinguished (36–159 for T7 DNA ligase and 30–104 for the Chlorella virus DNA ligase, Fig. 1, shown in blue), which contains a mobile loop, invisible in the crystal structure. Domain 1 contains four spatially conserved positively charged residues (Fig. 1, blue) that are proposed to interact with the 5¢-phosphate moiety of the nick [19,25]. Two of them, Lys238 and Lys240 of T7 DNA ligase (Lys188 and to a lesser extent Lys186 of Chlorella virus DNA ligase) were shown to be essential for the transadenylation and nick sealing activities [25,26]. Lys240 forms a photo-crosslinking adduct with the 5¢-terminal nucleotide of the nick, implying its direct involvement in binding of the nick phosphate [25]. Domain 1 contains the catalytic lysine residue that forms a covalent intermediate with the nucleotide coenzyme. The C-terminal domain of DNA ligase, domain 2 (Fig. 1, yellow), is smaller and is connected to domain 1 via the conserved motif D [25] (Fig. 1, red, in alternative notation called as motif V [27]). Domain 2 is also referred to as the OB (oligonucleotide/oligosaccharide binding)-fold domain, similar to those found in other DNA and RNA binding proteins [28,29]. Domain 2 is flexible; it was shown for the related nucleotidyltransferase, the mRNA capping enzyme from Chlorella virus PBCV-1, that during catalysis the enzyme undergoes opened–closed conformational changes upon which domain 2 moves towards domain 1 and closes the nucleotide binding site [30]. For DNA ligases it was suggested that this motion is connected with binding of both ATP and nicked dsDNA [25]. As to ATP, closing of domain 2 was proposed to adjust the conformation of the b–c pyrophosphate of ATP to a position favorable for the in-line nucleophilic attack of the catalytic lysyl moiety [19,23]. As to nicked dsDNA, closing of the domain 2 was proposed to clamp the enzyme on DNA [25]. dsDNA BINDING SITE Studies involving limited proteolysis, mutagenesis and molecular modeling strongly suggest that dsDNA binds ligase in the cleft between domains 1 and 2 [17,21,25,31–33]. Both domains of T7 DNA ligase bind dsDNA independ- ently, and, as expected, only domain 1 retains residual ligase activity [33]. On the basis of modeling studies it was shown that the motifs A and B of subdomain 1c, and C and D of domain 1 (Fig. 1, red) are involved in dsDNA binding [25]. It was suggested that the dsDNAÆprotein contacts traverse the whole of domain 1, and that the dsDNA binds right on top of the AMP bound in the active site [25,34]. The modeling did not elucidate a possible dsDNA-binding site of domain 2, perhaps because the opened conformation of the enzyme was used. Using DNA footprinting analysis it was shown that ligase binds nicked dsDNA asymmetrically, contacting 7–12 nucleotides at the 5¢-phosphate side of the nick, and 3–8 nucleotides at the 3¢-hydroxyl side [25,32,34]. With respect to the enzyme structure that would mean that motifs A and B must contact the 5¢-phosphate side of the nick of the dsDNA, because they are further away from the active site compared to motifs C and D [25]. STRUCTURAL SIMILARITY BETWEEN DNA LIGASE AND DNA POLYMERASE The catalytic core of DNA polymerase responsible for the dsDNA elongation activity contains three domains. Its shape resembles a half-opened hand (Fig. 2, left), and the domains are named accordingly [35,36]. The catalytic ÔpalmÕ domain contains the polymerase active site, where the incorporation of the nucleotide in the nascent primer chain takes place. dsDNA binds the palm domain in the cleft formed by the ÔthumbÕ and flexible ÔfingersÕ [37]. Similar to DNA ligase, DNA polymerase undergoes an opened–closed conformational change in the course of catalysis, upon which the fingers and the thumb domains close on the palm domain containing bound dsDNA and dNTP [37–41]. Fig. 1. Structure of T7 DNA ligase (left) and the DNA ligase–adenylylate complex from the Chlorella virus (right). Domain 1 is shown in green, subdomain 1c in blue and domain 2 in yellow. Motifs A–D are shown in red. Resi- dues that participate in binding of the nick phosphate are shown in blue. Some hydro- phobic residues in the putative DNA binding siteareshowninred.TheAMPmoietyof Chlorella virus DNA ligase adenylate is shown in purple. 5994 A. V. Cherepanov and S. de Vries (Eur. J. Biochem. 269) Ó FEBS 2002 Figure 2 shows that the analogy with a hand can be extended to DNA ligase. Domain 1 would be associated with the palm, subdomain 1c with the thumb and domain 2 with the flexible fingers. Also, the dsDNA binding mode proposed for DNA ligase in the modeling studies [25]. resembles the one of DNA polymerase (Fig. 2, left). dsDNA–POLYMERASE AND dsDNA–LIGASE INTERACTIONS The interaction of DNA polymerase with dsDNA is relatively well understood. In solution dsDNA generally prefers the B-type helix, but in the complex with polymerase up to 6 or 7 bp at the 3¢-end of the primer accommodate the A-form [41–43] (Fig. 2, left). One of the reasons for this is that the polymerase bends DNA, clamping the helix between the palm and the thumb domains [44–48]. A–B- form dsDNA hybrids are usually bent at the junction [49,50], so the induced bending by the protein stabilizes the A-form [42,51,52]. Another reason for the relative stability of A-form dsDNA in complex with the DNA polymerase is relatedtolessspecificdsDNAÆprotein interactions. They include (a) relatively high hydrophobicity of the dsDNA binding cleft compared to solution, which leads to a decrease of the degree of hydration of bound dsDNA, which stabilizes the A-type helix [53,54], and (b) replacement and/or exclusion of water molecules, which are normally hydrogen bonded to the dsDNA in solution, by the amino acid residues [55–57] and/or salt bridges [42] in the dsDNAÆprotein complex. The resulting effect can be compared with the addition of a hydrophobic solvent or with an increase of the ionic strength, factors which induce the B-to-A helix transition of dsDNA in solution [58,59]. In general, the induced B-to-A helix transition is a common feature of dsDNAÆprotein interactions [52,60–62], in par- ticular for the enzymes that catalyze sealing/cutting oper- ations on dsDNA [42]. It seems likely that the A-B dsDNA hybrid bend at the junction would fit DNA ligase better than the straight dsDNA, because the cleft between domains 1 and 2 is curved. In this case motif A of subdomain 1c (thumb) would contact the hybrid dsDNA at the A-B junction point, similar to the thumb–helix clamp motif of HIV-1 RT (Fig. 2, left). The distance between the junction point and the nick binding site is around 20 A ˚ , which corresponds to  7 bp of dsDNA. There are several aromatic residues in the active site of DNA ligase, which could stabilize the A-helix by hydrophobic and/or aromatic–aromatic interac- tions. Surprisingly, most of them are aligned parallel to each other along the putative dsDNA binding site (Fig. 1, red). DNA ligase undergoes an opened–closed conformational change during catalysis, which could stabilize the A-DNA motif by water exclusion and additional protein–DNA interactions. The pyrophosphate-bridging riboadenosine at the 5¢-end of the dsDNA nick might stimulate the B-to-A DNA conversion as well because of the structural influence of ribose sugar, similar to the other cases of A-DNA duplexes that contain a single ribose residue [63–65]. If the 7 bp-long fragment of bound dsDNA would adopt the A-form conformation in DNA ligase complex, it would cause an overall DNA unwinding of  20–25 degrees, because A-DNA contains roughly one more bp per turn of the helix than the B-form. It was shown that both ATP- and NAD + -dependent DNA ligases unwind dsDNA at the binding site at least for 17–20 degrees per bound molecule of theenzyme[66]. Therefore, there are sufficient grounds to suggest that the 6–9 bp-long B-DNA, at the 5¢-phosphate side of the nick, changes to A-DNA after formation of the DNA–ligase complex, similarly to the motif at the 3¢-OH primer end of the dsDNA bound to DNA polymerase. What could be the role of this transition in the DNA ligase catalysis? DYNAMIC MECHANISM OF NICK RECOGNITION BY DNA LIGASE – AHYPOTHESIS According to Doherty et al. [25,34], the adenylylated DNA ligase binds dsDNA forming nonspecific contacts with motifs A, B, C and D. According to our hypothesis the enzyme, in addition, bends dsDNA at the point of contact with motif A. Subdomain 1c, similar to the thumb domain of DNA polymerase, clamps on dsDNA bound in the crevice formed by domain 1 (palm) and domain 2 (fingers). This could be achieved by moving the tip (motif B) of subdomain 1c (thumb) towards domains 1 (palm), 2 (fingers) and bound dsDNA (Fig. 2, right, white arrow), similar to the motion of the thumb domain in DNA polymerases [35,37,41]. Nonspecific interactions lead to a decrease of the degree of hydration of the bound DNA. As a Fig. 2. Structures of DNA polymerase domain of HIV-1 reverse transcriptase in complex with dsDNA (left), and T7 DNA ligase (right). The connection domain of HIV-1 RT is omitted from the figure for clarity. The palm domain is showningreen,thethumbdomaininblueand the fingers domain in yellow. Directions of the catalytic movement of the thumb and fingers domains are indicated with white arrows. Ó FEBS 2002 Nick recognition by DNA ligase (Eur. J. Biochem. 269) 5995 result, the 6–9 bp dsDNA fragment between motifs A and C changes to the A-form helix. This transition is accom- panied by a dsDNA contraction of 5–7 A ˚ ,becausethe distance between the neighboring nucleotides is 2.6 A ˚ in the A-form vs. 3.4 A ˚ in the B-form. Contraction causes dsDNA to slip in the active site towards the clamp site (motif A) (Fig. 3). It also causes an overall DNA unwinding for 20–30 degrees (6 bp A-DNA contains  0.54 bp more per turn of a helix compared to 6 bp B-DNA, which corresponds to 360 degrees · 0.54/10–20 degrees unwinding angle). It is known that the 5¢-nick phosphate is essential for the tight binding of dsDNA by the DNA ligase. Several residues (Fig. 1, blue), which are located in domain 1 close to the bound AMP, are thought to bind to this moiety [19,25]. We propose that DNA ligase makes a two-force- point contact with nicked dsDNA – at the clamp site via motifAandatthe5¢-phosphate of the nick via the specific phosphate-binding residue(s) (e.g. Lys238 and Lys240 for T7 DNA ligase [19,25], or Arg42, Arg176 and Lys186 for Chlorella virus ligase [19,26]). At the clamp site, both DNA strands are fixed with respect to the enzyme, because DNA bends here. At the nick, however, only the 5¢-phosphate of the nicked strand is enzyme-bound (Fig. 3, ds nicked DNA). During the contraction, or B-to-A DNA helix transition, the non-nicked strand tends to adopt the A-DNA conformation, because it is anchored to the enzyme only at the clamp site and is free to slip in the active site. According to our hypothesis, the nicked strand has less freedom of conformational changes because it is anchored to DNA ligase at two points. Two extreme cases can be considered. The first case represents an enzyme, which would be structurally infinitely flexible between the two force points. In this case, contraction of dsDNA would drag the residues bound to the 5¢-phosphate of the nick several angstroms towards motif A. Some of these residues (e.g. Lys238 and Lys240 for T7 DNA ligase or Lys186 and Lys188 for Chlorella virus ligase) belong to motif D. This motif connects domains 1 and 2, and serves as a ÔhingeÕ during the opened–closed conformational change. So, the nick phosphate of dsDNA could pull on this hinge during contraction, triggering the closing of domain 2, and could further stall the ligase in the closed conformation until the nick is sealed. The other extreme case would be that the enzyme is structurally infinitely rigid between motif A and the nick phosphate-binding residue(s). In this case, the nicked strand wouldtendtoretainitsB-form,sinceitisfixedbothatthe clamp site and at the 5¢-phosphate of the nick. As a result, the DNA motif between the clamp site and the nick phosphate would adopt a strained hybrid conformation, in which the non-nicked strand is more A-like, while the nicked strand is more B-like (Fig. 3, nicked dsDNA, closed enzyme). One of the options for DNA to retain the hydrogen bonding of the 3¢-terminal base pair of the nick would be to slightly rotate counterclockwise around the helical axis, so that the 3¢-OH moiety would move towards the 5¢-phosphate of the nick and forward in the 3¢-direction of the nicked strand (Fig. 3, nicked dsDNA, closed enzyme). In other words, the 5¢-phosphate would move towards the protein interface, while the 3¢-OH group would move towards the solution. In this way, the 3¢-OH group would adopt the apical configuration in respect to the a-phosphorus moiety of the AMP cofactor (Fig. 4). For comparison, the nicked dsDNA bound to the DNA polymerase b makes a similar motion (for structure cf [39]), only in that case the 3¢-OH side of the nick moves away from the 5¢-phosphate of the nick and backwards in the 5¢-direction of the nicked strand. Fig. 3. Illustration of the proposed mechanism of dynamic nick recognition by DNA ligase. (Left) binding of the dsDNA. (Right) binding of the nicked dsDNA. B-form DNA is colored yellow, A-DNA is blue and the enzyme is showningreen. Fig. 4. Illustration of the proposed mechanism of dynamic nick recog- nition by DNA ligase. Positioning of the reacting groups in the active site of the enzyme for the B-DNA configuration and for the A–B strained DNA hybrid. 5996 A. V. Cherepanov and S. de Vries (Eur. J. Biochem. 269) Ó FEBS 2002 Most probably, the flexibility of DNA ligase is somewhere between these two extreme cases, so that the B-to-A helix transition of dsDNA would cause both the closure of domain 2 and the formation of the strained A–B configuration. In summary, if our hypothesis of dynamic nick recog- nition proves to be correct, DNA ligase would be a good example of an enzyme that acts according to induced-fit and strain mechanisms of catalysis in which both the enzyme and the substrate undergo significant conforma- tional changes to achieve the transition state configuration [67–69]. BINDING OF dsDNA TO DNA LIGASES It is important to note that the binding of dsDNA to the ligase is still a matter of some controversy. The results based on the electrophoretic mobility shift assay (EMSA) indicate that the ligase does not bind the non-nicked dsDNA, or dsDNA containing the nonphosphorylated nick [25,32,70]. On the other hand, other experiments that show relaxation of supercoiled DNA in the presence of T4 DNA ligase imply that the enzyme not only binds but also unwinds the non- nicked DNA helix [66]. In our opinion, the reason for this paradox is that the EMSA fails to detect proteinDNA complexes with k off values comparable to the apparent rate constant for diffusion of the proteinDNA complex through the pore of the acrylamide gel, k diff app .Fora5%gelthe apparent pore diameter is around 100–200 nm, depending on the bisacrylamide content [71]. Thus, for a 2 h separation with an electrophoretic shift of, for example, 5 cm, the k diff app can be estimated as 5 · 10 )2 m/200 · 10 )9 m ¼ 2.5 · 10 5 pores per 2 h, or 35–70 s )1 . This implies that only the complexes with k off of about 1–2 s )1 would be detected using this method. On the other hand, more rapidly exchanging complexes can be detected in the assay showing relaxation of the supercoiled DNA. B-TO-A DNA HELIX TRANSITION – A DYNAMICTESTOFTHESTATEOFTHE DNA SUBSTRATE We propose that the B-to-A DNA helix transition serves as a dynamic test to determine the state of the DNA substrate, and is used by DNA ligase to comply with its fidelity requirements. (A) To test for the presence of mismatching nucleotide(s) at the 3¢-hydroxyl side of the nick. Even though the A-B strained conformation can be adopted, the dangling 3¢-OH end would not occupy the position apical towards the leaving AMP, preventing the sealing of the nick, and, possibly, hindering the preceding transadenylation. This agreeswiththefactthatmismatchesatthe3¢-OH side of the nick in some cases inhibit not only the nick sealing activity [72], but adenylylation as well [73]. (B) To lower the large sequence-dependent structural variations at the 5¢-PO 4 side of the nick, and to test for the presence of mismatching nucleotide(s). The A-form of DNA is known to obey Ôstructural conservatismÕ, being rather independent of the primary sequence [74]. The presence of mismatches at the 5¢-PO 4 side of the nick destabilizes the A-form helix increasing the DNA hydration, because the water molecules tend to cluster around unusual base pairs to compensate for the absent hydrogen bonds [75]. (C) To test for the presence of an RNA motif at the 5¢-end of the nick. This important fidelity requirement would preclude DNA ligase to join the Okazaki fragments that contain RNA primer fragments, before they are removed by the 5¢)3¢-exonuclease activity of DNA polymerase [76] or by the action of specific RNases [77]. The B-to-A helix transition would not occur in case of the nick containing 5¢-RNAÆDNA, because the RNAÆDNA hybrid already adopts the A-like form in solution. As a result, the A-B strained conformation would not be achieved, the 3¢-OH group of the nick would not occupy the position apical to the leaving AMP and the nick sealing would be inhibited. The latter agrees with the fact that the DNA ligase joins 5¢-RNAÆDNA to the 3¢-DNAÆDNA poorly, leading to the accumulation of the DNA-adenylate intermediate, while the opposite situation results in effective ligation [78,79]. It is necessary to note, however, that in certain cases DNA ligase is capable of joining nicks containing the RNA/DNA motif on the 5¢-side with reduced efficiency [72,79–82]. In these cases, generally, oligo-d(r)A/oligo-r(d)T sequences were ligated, which, for dsDNA, have a very low tendency to form the A-helix in solution [83–85]. The A-helix is not the only possible conformation of the DNAÆRNA chimera; sometimes it rather adopts a mixed A–B-geometry [86,87], or, under certain conditions, even the B-helical conforma- tion [88]. Therefore, it is possible that the oligo-d(r)A/oligo- r(d)T DNAÆRNA hybrids in solution adopt the B-like structure, and in complex with DNA ligase undergo a B-to-A helix transition, allowing nick-joining. In summary, we propose that DNA ligase transiently probes dsDNA by bending the DNA helix, unwinding, and inducing the B-to-A helix transition. A defect in the DNA helix, such as a phosphorylated nick reveals itself during the dynamic test, forcing (a) DNA ligase to form a stable complex with dsDNA by changing to a closed conforma- tion, and (b) dsDNA to adopt a conformation favorable for the transadenylation and sealing of the nick. ACKNOWLEDGEMENTS This work was supported by Association Of Biotechnology Centers In the Netherlands (ABON) (Project I.2.8) and by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW) (grant 349–3565). REFERENCES 1. Lehman, I.R. (1974) DNA ligase: structure, mechanism, and function. 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Nat Struct. Biol. 2, 733–735. Ó FEBS 2002 Nick recognition by DNA ligase (Eur. J. Biochem. 269) 5999 . of the dsDNA bound to DNA polymerase. What could be the role of this transition in the DNA ligase catalysis? DYNAMIC MECHANISM OF NICK RECOGNITION BY DNA LIGASE. ARTICLE Dynamic mechanism of nick recognition by DNA ligase Alexei V. Cherepanov* and Simon de Vries Kluyver Department of Biotechnology, Delft University of