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DNA glycosylase recognition and catalysis
DNA glycosylase recognition and catalysis J Christopher Fromme1, Anirban Banerjee2 and Gregory L Verdine1,2, DNA glycosylases are the enzymes responsible for recognizing base lesions in the genome and initiating base excision DNA repair. Recent structural and biochemical results have provided novel insights into DNA damage recognition and repair. The basis of the recognition of the oxidative lesion 8-oxoguanine by two structurally unrelated DNA glycosylases is now understood and has been revealed to involve surprisingly similar strategies. Work on MutM (Fpg) has produced structures representing three discrete reaction steps. The NMR structure of 3-methyladenine glycosylase I revealed its place among the structural families of DNA glycosylases and the X-ray structure of SMUG1 likewise confirmed that this protein is a member of the uracil DNA glycosylase superfamily. A novel disulfide cross-linking strategy was used to obtain the long-anticipated structure of MutY bound to DNA containing an AoxoG mispair. Addresses Departments of 1Molecular and Cellular Biology, and 2Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA e-mail: [email protected]
normal DNA represents a formidable biological version of the ‘needle in the haystack’ problem. DNA glycosylases fall into two major classes, which differ with respect to catalytic mechanism. Monofunctional glycosylases catalyze the single-step displacement of the damaged base via attack of an activated water molecule on C10 of the substrate (Figure 1). Bifunctional DNA glycosylases also excise the lesion base, but, instead of water, use an amine nucleophile of the protein to attack C10 ; the covalently linked enzyme–substrate complex proceeds through a multistep reaction cascade leading to severance of the DNA backbone on the 30 side, followed in some cases by backbone severance on the 50 side as well (Figure 1). Interestingly, DNA glycosylases that share the same fold sometimes differ in their mechanistic class.
Introduction
Four structural superfamilies of DNA glycosylases have been identified thus far. Here, we refer to these as the UDG, AAG and MutM/Fpg superfamilies, based on structural similarity to uracil DNA glycosylase (UDG), alkyladenine DNA glycosylase (AAG) and bacterial 8oxoguanine DNA glycosylase (MutM/Fpg), respectively, and the HhH-GPD superfamily, named for a characteristic active site motif borne by family members comprising a helix-hairpin-helix followed by a Gly/Pro-rich loop and catalytic aspartate residue [2,3]. Members of the UDG and AAG superfamilies are compact single-domain enzymes with relatively small DNA-interaction surfaces. Proteins of the MutM/Fpg and HhH-GPD superfamilies contain multiple domains, with the active site located at an interdomain junction; these proteins often contain additional domains that serve specialized biological roles. The MutM/Fpg and HhH-GPD families are distinctive in that some family members possess structural metal ions or clusters. Although structures of members of the four superfamilies have no discernible overall similarity, all share the common feature of binding primarily to the lesion-containing DNA strand, with the lesion nucleobase being extruded from the duplex and inserted into an extrahelical recognition pocket on the enzyme.
Most damage to bases in DNA is repaired by the base excision repair (BER) pathway [1]. BER is initiated by DNA glycosylases, enzymes that recognize specific lesion bases in DNA and remove them. The resulting abasic lesion is then further processed by endonucleases, a repair polymerase and ligase, ultimately to effect complete restoration of the original DNA sequence. Each DNA glycosylase is programmed via its structure to recognize and act upon a particular cohort of lesions; locating these damaged bases embedded in a million-fold excess of
Tainer and co-workers [4] determined the first structure of a DNA glycosylase, namely that of the prototypical HhH-GPD family member endonuclease III (Nth). This structure provided vital information to a structurally impoverished DNA repair community, but also made it clear that structures of enzyme–DNA complexes would be required before sound conclusions could be made with regard to lesion recognition and catalysis. Thus, the goal of structural biologists working in this area has been to
Current Opinion in Structural Biology 2004, 14:43–49 This review comes from a themed issue on Protein–nucleic acid interactions Edited by Gregory D Van Duyne and Wei Yang 0959-440X/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2004.01.003
Abbreviations AAG alkyladenine DNA glycosylase BER base excision repair hOGG1 human 8-oxoguanine glycosylase 1 oxoG 8-oxoguanine PDB Protein Data Bank TDG thymine DNA glycosylase UDG uracil DNA glycosylase
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Current Opinion in Structural Biology 2004, 14:43–49
44 Protein–nucleic acid interactions
Figure 1
DNA -O O
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Mechanistic scheme of the reactions catalyzed by DNA glycosylases. DNA glycosylases must first recognize and bind to nucleobase damage (1). Monofunctional DNA glycosylases, such as UDG and MutY, then catalyze the one-step removal of the lesion base to generate an abasic site (4). Bifunctional DNA glycosylases, such as MutM and EndoIII, catalyze additional reactions via the attachment of an active site nucleophilic moiety (Nuc) (2), resulting in 30 (3; e.g. EndoIII), or 30 and 50 (6; e.g. MutM) nicking of the DNA strand. A characteristic of the Schiff base intermediate (2) is that it can be intercepted by the reducing agent sodium borohydride (NaBH4) to generate a stable covalent enzyme–DNA complex (5).
obtain co-crystal structures representing each discrete step along the reaction pathway, from lesion recognition to product release. When the field was last reviewed in 2001 [5,6], only a handful of structures were available, mostly of damage-recognition complexes. Tremendous progress has been made in the short time since and, as discussed below, recent structures have greatly expanded our understanding of lesion recognition and catalysis.
MutM/Fpg family All known members of the MutM/Fpg family of DNA glycosylases have the unique and surprising feature of using their N-terminal proline residue as the key catalytic nucleophile. Great strides have been made recently toward characterizing the multifaceted functions of this structural family. Of particular note are studies on Bacillus stearothermophilus MutM (Figure 2a), which have provided structures representing several critical stages of the repair process: the precatalytic lesion-recognition complex, an intermediate in the middle of the reaction cascade and the end product of lesion processing (Figure 3) [7,8]. Independently determined structures of repair intermediates bound to MutM from other organisms [9–11] and of the orthologous protein endonuclease VIII (EndoVIII, Nei gene) [11,12] are virtually identical to the corresponding B. stearothermophilus structures, thus providing mutual validation of the conclusions. These Current Opinion in Structural Biology 2004, 14:43–49
studies, taken together, represent the greatest advance made thus far toward the over-riding goal of obtaining a structural snapshot of each discrete reaction step catalyzed by a bifunctional DNA glycosylase. MutM uses a single arginine sidechain to recognize the base opposite the lesion (preferring cytosine) and holds the substrate lesion base in its active site through multiple interactions with the phosphate backbone. MutM (and presumably EndoVIII) possesses a flexible loop involved in lesion base recognition. Interestingly, this loop becomes disordered following removal of the substrate 8-oxoguanine (oxoG) lesion base — as observed in structures of the trapped intermediate and end product DNA complexes [7,9] — but is ordered in the absence of any DNA [13]. The mode of oxoG recognition by MutM is similar to that of the structurally unrelated enzyme human 8-oxoguanine glycosylase 1 (hOGG1), relying on the protonation state of N7 of oxoG (N7 bears a proton in oxoG, but not in guanine). However, the oxoG nucleoside is bound in the syn conformation to the MutM active site, in contrast to the anti conformation adopted when bound to hOGG1 [14]. The structure of MutM bound to DNA containing an alternative lesion base, dihydrouracil, demonstrates the way in which MutM is able to repair different lesions by recognizing similar patterns of hydrogen-bond donors and acceptors on lesion bases [8]. www.sciencedirect.com
DNA glycosylases Fromme, Banerjee and Verdine 45
Figure 2
(a)
(b)
(c)
Current Opinion in Structural Biology
Structures of several recently determined DNA glycosylase–DNA complexes. The DNA is shown as a gold tube model, the lesion is shown in red ball-and-stick representation, the base opposite the lesion is shown in black ball-and-stick representation and the protein ribbon is colored by domain. (a) Structure of MutM bound to DNA containing an oxoGC pair (PDB code 1R2Y [8]). The structurally important zinc ion is shown as a green sphere. (b) The HhH-GPD superfamily member EndoIII bound to DNA containing an abasic site (PDB code 1P59 [38]). The [4Fe–4S] cluster can be seen as orange and yellow spheres. (c) SMUG1–DNA complex (PDB code 1OE5 [51]). The lesion has adopted two conformations and has actually been processed in one of the conformations. Figures 2–4 prepared with RIBBONS [52].
The work on EndoVIII is of special importance because, to date, it is the only family member for which a structure is available for both the DNA-bound and unbound forms of the enzyme from the same organism (Escherichia coli). Comparison of the bound and unbound forms revealed that DNA binding is accompanied by a slight (58) hinge motion at the interface between the two protein domains, which allows the protein to tighten its hold on the duplex [11]. However, this degree of motion is modest in comparison to the large domain shifts seen, for example, in protein kinases. The relatively rigid domain structure of DNA glycosylases may be necessary to induce drastic remodeling of their DNA substrates. Believed for some time to be restricted phylogenetically to prokaryotes, MutM orthologs in higher eukaryotes have recently been identified and preliminarily characterized [15–19]. High-resolution structural information is not yet available on these so-called NEIL (for Nei-like) proteins.
HhH-GPD superfamily Members of the HhH-GPD superfamily repair diverse lesions, including oxidative, alkylation and hydrolytic damage, through either monofunctional or bifunctional mechanisms. Early structural studies of hOGG1 and AlkA established the general principles of DNA lesion recognition by family members [14,20]. The crystal structure of hOGG1 in the absence of DNA revealed some subtle changes in backbone and sidechain positions that accompany DNA binding [21]. More recent work has elucidated mechanistic details of the reaction catalyzed by hOGG1, through a combination of structural and biochemical www.sciencedirect.com
studies [22,23]. A particularly interesting aspect of the hOGG1 mechanism is that the product of base excision — oxoG free base — remains bound in the enzyme active site and acts as an acid/base cofactor to accelerate the rate of the downstream strand-nicking reaction; such productassisted catalysis has never before been seen in any enzyme-catalyzed reaction. NMR structural studies led to the identification of constitutive E. coli 3-methyladenine glycosylase I (MagI) as a novel zinc-containing HhH family member [24,25]. The structure revealed an unusual architecture, which appears to be lacking the otherwise absolutely conserved GPD motif, including its key catalytic aspartate residue. The NMR structure of MagI bound to 3-methyladenine, combined with a fluorescence-based investigation of lesion analog binding, suggested that discrimination for methylated adenine occurs primarily through van der Waals interactions [26]. The crystal structure of 3methyladenine glycosylase III (MagIII) in complex with two different alkylated adenine free base analogs and accompanying biochemical work demonstrated that the source of MagIII discrimination against an alternative methylation product, 7-methyladenine, is steric exclusion [27]. In contrast to MagI, which uses hydrogen bonds to recognize 3-methyladenine, MagIII appears to rely entirely on aromatic stacking for recognition of lesion bases. Interestingly, mutation of the catalytic aspartate residue to asparagine was found to diminish, but not abolish, MagIII catalytic activity [27]. TG mismatches can arise in eukaryotic genomes through deamination of methylated cytosine residues in CpG Current Opinion in Structural Biology 2004, 14:43–49
46 Protein–nucleic acid interactions
Figure 3
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βF-α10 Loop Tyr242
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sites [30]. The crystal structure of this domain confirmed its place within the HhH-GPD superfamily, and led to a proposed mechanism for thymine recognition based on a specific combination of hydrogen bonds and van der Waals forces [31]. Bacteria also possess a HhH-GPD TG glycosylase, presumably for maintenance of methylated restriction sites, named Mig [32]. Mig is structurally unrelated to TDG (thymine DNA glycosylase), the predominant TG glycosylase in eukaryotes and a member of the UDG structural family. The recently determined structure of Mig demonstrated high structural similarity to other HhH-GPD family members. Substrate modeling led to the hypothesis that this enzyme uses strain energy as the primary driving force for catalysis [33]. Mig contains a structural [4Fe–4S] cluster, as do two other HhH-GPD family members: endonuclease III (EndoIII) and MutY. EndoIII, Mig and the N-terminal domain of MutY share high structural homology [33], with EndoIII and the N-terminal domain of MutY having 31% identity in E. coli. MutY additionally possesses a C-terminal domain, which has been shown to be structurally homologous to MutT, an enzyme responsible for hydrolyzing 8-oxo-dGTP [34,35]. This architectural design is logical, considering that the function of MutY is to remove adenine from AoxoG mispairs. The role of the [4Fe– 4S] cluster in HhH-GPD glycosylases has remained unclear. Earlier studies demonstrated that the iron–sulfur cluster of EndoIII was redox inert [36], but a very recent study using DNA-modified electrodes determined that the MutY [4Fe–4S] cluster is redox active when the enzyme is bound to DNA [37]. The authors of this work suggest that the redox state of the cluster could modulate the affinity of MutY for DNA; thus, the redox activity of the cluster may play a role in searching for damage.
Pro2
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Current Opinion in Structural Biology
Structural snapshots of catalysis by MutM. DNA is colored gold, with the oxoG lesion nucleoside in red. Amino acid sidechains are colored cyan. The protein backbone is shown as a gray ribbon, except for the flexible bF-a10 loop, colored purple. Hydrogen bonds are shown as dashed lines with distances. (a) The active site of the recognition complex (PDB code 1R2Y [8]). Note the syn disposition of the glycosidic bond. (b) The active site of the borohydride-trapped intermediate (PDB code 1L1Z [7]). Water molecules are visible as red spheres. The reduced Schiff base bond is indicated with a purple arrow. (c) The active site of the MutM–product DNA complex (PDB code 1L2B [7]).
islands [28,29]. The C-terminal domain of the methylCpG-binding protein MBD4 is actually a DNA glycosylase that removes thymine from TG pairs at methyl-CpG Current Opinion in Structural Biology 2004, 14:43–49
The recent structure determination of a covalently trapped EndoIII–DNA intermediate (Figure 2b) is the first for any [4Fe–4S] enzyme bound to DNA [38]. The structure provided no evidence of a direct role for the cluster in damage recognition, but did point to an indirect structural role in orienting a loop of poorly conserved residues for interaction with the DNA duplex distal to the site of damage. An unusual aspect of the EndoIII–DNA complex is the extensive number of contacts the enzyme makes with the strand of DNA complementary to the one containing the lesion. The structure also provided an atomic basis for understanding the preference of EndoIII to repair lesions paired opposite guanine residues in DNA; the enzyme uses mainchain carbonyl oxygens to recognize the Watson–Crick face of the guanine opposite the lesion. A unique and interesting feature of MutY is that it removes a normal adenine base when paired opposite the mutagenic lesion oxoG. The structural basis of the recognition of a lesion outside of the MutY active site www.sciencedirect.com
DNA glycosylases Fromme, Banerjee and Verdine 47
Figure 4
C-terminal domain
Catalytic domain
oxoG
N
Adenine C Current Opinion in Structural Biology
Structure of MutY bound to DNA containing an AoxoG pair (PDB code 1RRQ [39]). DNA is shown in gold CPK format, with the oxoG base colored red and the adenine base colored purple. MutY is shown in ribbon format with termini labeled. The C-terminal domain, on the left, is responsible for oxoG recognition. The [4Fe–4S] cluster and a calcium ion are shown as spheres.
has been highly anticipated. The structure of MutY in complex with DNA containing an AoxoG mispair (Figure 4) was recently determined [39] with the aid of disulfide cross-linking [40]. The enzyme recognizes oxoG using hydrogen bonds by sandwiching the oxoGcontaining strand of DNA between the N- and C-terminal domains. The oxoG residue remains intrahelical, but is bound in the anti glycosidic conformation, in contrast to the syn conformation found when paired with adenine in DNA. This observation impacts hypotheses concerning both the search mechanism and the binding events preceding catalysis. The structure also provides a clear explanation for the deleterious effects of two MutY polymorphisms found in some hereditary colon cancers, as these mutations interfere with the faithful recognition of oxoG.
UDG family UDG (or Ung) is probably the most widely studied DNA glycosylase and the structural biology of repair by UDG (and the homologous enzyme Mug) is well developed [41–44]. Work on the structural biology and monofunctional mechanism of UDG continues to bear valuable fruit. Structural studies of UDG bound to an inhibitor [45], together with biochemical studies [46], suggest that electrostatic interactions and geometric strain play large roles in catalysis. Additional evidence has demonstrated the electrostatic contribution of phosphates in the DNA backbone to catalysis [47,48] and indicates that base excision occurs through a dissociative SN1-type mechanism [48,49]. Using the structure as a guide, a mutant form of UDG was engineered, via the introduction of two amino acid substitutions, to exhibit alternative specificity for cytosine when paired opposite bulky lesions [50]. www.sciencedirect.com
The determination of the crystal structure of a SMUG1– DNA complex [51] (Figure 2c) established conclusively that this protein is indeed a member of the UDG superfamily, despite having less than 10% sequence identity with any other known member. The structures of several different SMUG1–DNA complexes were determined, representing product and nonspecific DNA complexes. The mechanism of the exclusion of thymine as a substrate is proposed to be based on the presence of an ordered water molecule in the base-recognition pocket, and the interaction of the enzyme with DNA appears to be more invasive into the duplex than seen with UDG. Further studies on trapped complexes will be necessary to test this notion.
Conclusions The structural biology of DNA glycosylases has progressed rapidly in the past few years. With attention centering on complexes between DNA glycosylases and DNA, the details of lesion recognition and catalysis are coming into focus. However, many interesting questions remain to be answered. For example, despite work on MutM that has yielded structures of three distinct stages of catalysis, the catalytic mechanism remains poorly understood. Future studies will continue to bring to light the structural basis of recognition and catalysis, but the next frontier in DNA glycosylase biology concerns the nature of the damage search process. How DNA glycosylases locate damaged bases present at very low levels in the genome has remained a mystery. We are hopeful that structures of DNA glycosylases bound to undamaged DNA will begin to flush out certain aspects of the search mechanism.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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48 Protein–nucleic acid interactions
Crystal structures of MutM–DNA complexes provide snapshots of a catalytic intermediate and the product, as well as insights into recognition of the base paired opposite the lesion.
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DNA glycosylases Fromme, Banerjee and Verdine 49
The crystal structure of MutY bound to AoxoG-containing DNA was obtained with the use of disulfide cross-linking. The structure reveals an intrahelical oxoG recognition mode in which the oxoG base has rotated about its glycosidic bond.
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48. Dinner AR, Blackburn GM, Karplus M: Uracil-DNA glycosylase acts by substrate autocatalysis. Nature 2001, 413:752-755.
43. Mol CD, Arvai AS, Slupphaug G, Kavli B, Alseth I, Krokan HE, Tainer JA: Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell 1995, 80:869-878. 44. Barrett TE, Savva R, Panayotou G, Barlow T, Brown T, Jiricny J, Pearl LH: Crystal structure of a G:T/U mismatch-specific DNA glycosylase: mismatch recognition by complementary-strand interactions. Cell 1998, 92:117-129. 45. Bianchet MA, Seiple LA, Jiang YL, Ichikawa Y, Amzel LM, Stivers JT: Electrostatic guidance of glycosyl cation migration
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49. Werner RM, Stivers JT: Kinetic isotope effect studies of the reaction catalyzed by uracil DNA glycosylase: evidence for an oxocarbenium ion-uracil anion intermediate. Biochemistry 2000, 39:14054-14064. 50. Kwon K, Jiang YL, Stivers JT: Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair. Chem Biol 2003, 10:351-359. 51. Wibley JE, Waters TR, Haushalter K, Verdine GL, Pearl LH: Structure and specificity of the vertebrate anti-mutator uracil-DNA glycosylase SMUG1. Mol Cell 2003, 11:1647-1659. 52. Carson M: Ribbons. Methods Enzymol 1997, 277:493-505.
Current Opinion in Structural Biology 2004, 14:43–49
normal DNA represents a formidable biological version of the ‘needle in the haystack’ problem. DNA glycosylases fall into two major classes, which differ with respect to catalytic mechanism. Monofunctional glycosylases catalyze the single-step displacement of the damaged base via attack of an activated water molecule on C10 of the substrate (Figure 1). Bifunctional DNA glycosylases also excise the lesion base, but, instead of water, use an amine nucleophile of the protein to attack C10 ; the covalently linked enzyme–substrate complex proceeds through a multistep reaction cascade leading to severance of the DNA backbone on the 30 side, followed in some cases by backbone severance on the 50 side as well (Figure 1). Interestingly, DNA glycosylases that share the same fold sometimes differ in their mechanistic class.
Introduction
Four structural superfamilies of DNA glycosylases have been identified thus far. Here, we refer to these as the UDG, AAG and MutM/Fpg superfamilies, based on structural similarity to uracil DNA glycosylase (UDG), alkyladenine DNA glycosylase (AAG) and bacterial 8oxoguanine DNA glycosylase (MutM/Fpg), respectively, and the HhH-GPD superfamily, named for a characteristic active site motif borne by family members comprising a helix-hairpin-helix followed by a Gly/Pro-rich loop and catalytic aspartate residue [2,3]. Members of the UDG and AAG superfamilies are compact single-domain enzymes with relatively small DNA-interaction surfaces. Proteins of the MutM/Fpg and HhH-GPD superfamilies contain multiple domains, with the active site located at an interdomain junction; these proteins often contain additional domains that serve specialized biological roles. The MutM/Fpg and HhH-GPD families are distinctive in that some family members possess structural metal ions or clusters. Although structures of members of the four superfamilies have no discernible overall similarity, all share the common feature of binding primarily to the lesion-containing DNA strand, with the lesion nucleobase being extruded from the duplex and inserted into an extrahelical recognition pocket on the enzyme.
Most damage to bases in DNA is repaired by the base excision repair (BER) pathway [1]. BER is initiated by DNA glycosylases, enzymes that recognize specific lesion bases in DNA and remove them. The resulting abasic lesion is then further processed by endonucleases, a repair polymerase and ligase, ultimately to effect complete restoration of the original DNA sequence. Each DNA glycosylase is programmed via its structure to recognize and act upon a particular cohort of lesions; locating these damaged bases embedded in a million-fold excess of
Tainer and co-workers [4] determined the first structure of a DNA glycosylase, namely that of the prototypical HhH-GPD family member endonuclease III (Nth). This structure provided vital information to a structurally impoverished DNA repair community, but also made it clear that structures of enzyme–DNA complexes would be required before sound conclusions could be made with regard to lesion recognition and catalysis. Thus, the goal of structural biologists working in this area has been to
Current Opinion in Structural Biology 2004, 14:43–49 This review comes from a themed issue on Protein–nucleic acid interactions Edited by Gregory D Van Duyne and Wei Yang 0959-440X/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2004.01.003
Abbreviations AAG alkyladenine DNA glycosylase BER base excision repair hOGG1 human 8-oxoguanine glycosylase 1 oxoG 8-oxoguanine PDB Protein Data Bank TDG thymine DNA glycosylase UDG uracil DNA glycosylase
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Current Opinion in Structural Biology 2004, 14:43–49
44 Protein–nucleic acid interactions
Figure 1
DNA -O O
Lesion base
P
–
O
O
O –
–
O
DNA-O
O
–O
P O
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1′
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O OH 1′
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–O
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O P O
.. Nuc DNA glycosylase
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..
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O P
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–
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–O
..
O P
O P
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..
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3′- and 5′-cleavage product (6) Current Opinion in Structural Biology
Mechanistic scheme of the reactions catalyzed by DNA glycosylases. DNA glycosylases must first recognize and bind to nucleobase damage (1). Monofunctional DNA glycosylases, such as UDG and MutY, then catalyze the one-step removal of the lesion base to generate an abasic site (4). Bifunctional DNA glycosylases, such as MutM and EndoIII, catalyze additional reactions via the attachment of an active site nucleophilic moiety (Nuc) (2), resulting in 30 (3; e.g. EndoIII), or 30 and 50 (6; e.g. MutM) nicking of the DNA strand. A characteristic of the Schiff base intermediate (2) is that it can be intercepted by the reducing agent sodium borohydride (NaBH4) to generate a stable covalent enzyme–DNA complex (5).
obtain co-crystal structures representing each discrete step along the reaction pathway, from lesion recognition to product release. When the field was last reviewed in 2001 [5,6], only a handful of structures were available, mostly of damage-recognition complexes. Tremendous progress has been made in the short time since and, as discussed below, recent structures have greatly expanded our understanding of lesion recognition and catalysis.
MutM/Fpg family All known members of the MutM/Fpg family of DNA glycosylases have the unique and surprising feature of using their N-terminal proline residue as the key catalytic nucleophile. Great strides have been made recently toward characterizing the multifaceted functions of this structural family. Of particular note are studies on Bacillus stearothermophilus MutM (Figure 2a), which have provided structures representing several critical stages of the repair process: the precatalytic lesion-recognition complex, an intermediate in the middle of the reaction cascade and the end product of lesion processing (Figure 3) [7,8]. Independently determined structures of repair intermediates bound to MutM from other organisms [9–11] and of the orthologous protein endonuclease VIII (EndoVIII, Nei gene) [11,12] are virtually identical to the corresponding B. stearothermophilus structures, thus providing mutual validation of the conclusions. These Current Opinion in Structural Biology 2004, 14:43–49
studies, taken together, represent the greatest advance made thus far toward the over-riding goal of obtaining a structural snapshot of each discrete reaction step catalyzed by a bifunctional DNA glycosylase. MutM uses a single arginine sidechain to recognize the base opposite the lesion (preferring cytosine) and holds the substrate lesion base in its active site through multiple interactions with the phosphate backbone. MutM (and presumably EndoVIII) possesses a flexible loop involved in lesion base recognition. Interestingly, this loop becomes disordered following removal of the substrate 8-oxoguanine (oxoG) lesion base — as observed in structures of the trapped intermediate and end product DNA complexes [7,9] — but is ordered in the absence of any DNA [13]. The mode of oxoG recognition by MutM is similar to that of the structurally unrelated enzyme human 8-oxoguanine glycosylase 1 (hOGG1), relying on the protonation state of N7 of oxoG (N7 bears a proton in oxoG, but not in guanine). However, the oxoG nucleoside is bound in the syn conformation to the MutM active site, in contrast to the anti conformation adopted when bound to hOGG1 [14]. The structure of MutM bound to DNA containing an alternative lesion base, dihydrouracil, demonstrates the way in which MutM is able to repair different lesions by recognizing similar patterns of hydrogen-bond donors and acceptors on lesion bases [8]. www.sciencedirect.com
DNA glycosylases Fromme, Banerjee and Verdine 45
Figure 2
(a)
(b)
(c)
Current Opinion in Structural Biology
Structures of several recently determined DNA glycosylase–DNA complexes. The DNA is shown as a gold tube model, the lesion is shown in red ball-and-stick representation, the base opposite the lesion is shown in black ball-and-stick representation and the protein ribbon is colored by domain. (a) Structure of MutM bound to DNA containing an oxoGC pair (PDB code 1R2Y [8]). The structurally important zinc ion is shown as a green sphere. (b) The HhH-GPD superfamily member EndoIII bound to DNA containing an abasic site (PDB code 1P59 [38]). The [4Fe–4S] cluster can be seen as orange and yellow spheres. (c) SMUG1–DNA complex (PDB code 1OE5 [51]). The lesion has adopted two conformations and has actually been processed in one of the conformations. Figures 2–4 prepared with RIBBONS [52].
The work on EndoVIII is of special importance because, to date, it is the only family member for which a structure is available for both the DNA-bound and unbound forms of the enzyme from the same organism (Escherichia coli). Comparison of the bound and unbound forms revealed that DNA binding is accompanied by a slight (58) hinge motion at the interface between the two protein domains, which allows the protein to tighten its hold on the duplex [11]. However, this degree of motion is modest in comparison to the large domain shifts seen, for example, in protein kinases. The relatively rigid domain structure of DNA glycosylases may be necessary to induce drastic remodeling of their DNA substrates. Believed for some time to be restricted phylogenetically to prokaryotes, MutM orthologs in higher eukaryotes have recently been identified and preliminarily characterized [15–19]. High-resolution structural information is not yet available on these so-called NEIL (for Nei-like) proteins.
HhH-GPD superfamily Members of the HhH-GPD superfamily repair diverse lesions, including oxidative, alkylation and hydrolytic damage, through either monofunctional or bifunctional mechanisms. Early structural studies of hOGG1 and AlkA established the general principles of DNA lesion recognition by family members [14,20]. The crystal structure of hOGG1 in the absence of DNA revealed some subtle changes in backbone and sidechain positions that accompany DNA binding [21]. More recent work has elucidated mechanistic details of the reaction catalyzed by hOGG1, through a combination of structural and biochemical www.sciencedirect.com
studies [22,23]. A particularly interesting aspect of the hOGG1 mechanism is that the product of base excision — oxoG free base — remains bound in the enzyme active site and acts as an acid/base cofactor to accelerate the rate of the downstream strand-nicking reaction; such productassisted catalysis has never before been seen in any enzyme-catalyzed reaction. NMR structural studies led to the identification of constitutive E. coli 3-methyladenine glycosylase I (MagI) as a novel zinc-containing HhH family member [24,25]. The structure revealed an unusual architecture, which appears to be lacking the otherwise absolutely conserved GPD motif, including its key catalytic aspartate residue. The NMR structure of MagI bound to 3-methyladenine, combined with a fluorescence-based investigation of lesion analog binding, suggested that discrimination for methylated adenine occurs primarily through van der Waals interactions [26]. The crystal structure of 3methyladenine glycosylase III (MagIII) in complex with two different alkylated adenine free base analogs and accompanying biochemical work demonstrated that the source of MagIII discrimination against an alternative methylation product, 7-methyladenine, is steric exclusion [27]. In contrast to MagI, which uses hydrogen bonds to recognize 3-methyladenine, MagIII appears to rely entirely on aromatic stacking for recognition of lesion bases. Interestingly, mutation of the catalytic aspartate residue to asparagine was found to diminish, but not abolish, MagIII catalytic activity [27]. TG mismatches can arise in eukaryotic genomes through deamination of methylated cytosine residues in CpG Current Opinion in Structural Biology 2004, 14:43–49
46 Protein–nucleic acid interactions
Figure 3
(a)
βF-α10 Loop Tyr242
Glu6
oxoG
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3.0 Å 1′ Gln3 5′ 4′
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3.2 Å 2.6 Å
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(c) Tyr242 Glu6
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sites [30]. The crystal structure of this domain confirmed its place within the HhH-GPD superfamily, and led to a proposed mechanism for thymine recognition based on a specific combination of hydrogen bonds and van der Waals forces [31]. Bacteria also possess a HhH-GPD TG glycosylase, presumably for maintenance of methylated restriction sites, named Mig [32]. Mig is structurally unrelated to TDG (thymine DNA glycosylase), the predominant TG glycosylase in eukaryotes and a member of the UDG structural family. The recently determined structure of Mig demonstrated high structural similarity to other HhH-GPD family members. Substrate modeling led to the hypothesis that this enzyme uses strain energy as the primary driving force for catalysis [33]. Mig contains a structural [4Fe–4S] cluster, as do two other HhH-GPD family members: endonuclease III (EndoIII) and MutY. EndoIII, Mig and the N-terminal domain of MutY share high structural homology [33], with EndoIII and the N-terminal domain of MutY having 31% identity in E. coli. MutY additionally possesses a C-terminal domain, which has been shown to be structurally homologous to MutT, an enzyme responsible for hydrolyzing 8-oxo-dGTP [34,35]. This architectural design is logical, considering that the function of MutY is to remove adenine from AoxoG mispairs. The role of the [4Fe– 4S] cluster in HhH-GPD glycosylases has remained unclear. Earlier studies demonstrated that the iron–sulfur cluster of EndoIII was redox inert [36], but a very recent study using DNA-modified electrodes determined that the MutY [4Fe–4S] cluster is redox active when the enzyme is bound to DNA [37]. The authors of this work suggest that the redox state of the cluster could modulate the affinity of MutY for DNA; thus, the redox activity of the cluster may play a role in searching for damage.
Pro2
2.5 Å Arg264
3.2 Å
3.1 Å
Lys60
3.8 Å
Current Opinion in Structural Biology
Structural snapshots of catalysis by MutM. DNA is colored gold, with the oxoG lesion nucleoside in red. Amino acid sidechains are colored cyan. The protein backbone is shown as a gray ribbon, except for the flexible bF-a10 loop, colored purple. Hydrogen bonds are shown as dashed lines with distances. (a) The active site of the recognition complex (PDB code 1R2Y [8]). Note the syn disposition of the glycosidic bond. (b) The active site of the borohydride-trapped intermediate (PDB code 1L1Z [7]). Water molecules are visible as red spheres. The reduced Schiff base bond is indicated with a purple arrow. (c) The active site of the MutM–product DNA complex (PDB code 1L2B [7]).
islands [28,29]. The C-terminal domain of the methylCpG-binding protein MBD4 is actually a DNA glycosylase that removes thymine from TG pairs at methyl-CpG Current Opinion in Structural Biology 2004, 14:43–49
The recent structure determination of a covalently trapped EndoIII–DNA intermediate (Figure 2b) is the first for any [4Fe–4S] enzyme bound to DNA [38]. The structure provided no evidence of a direct role for the cluster in damage recognition, but did point to an indirect structural role in orienting a loop of poorly conserved residues for interaction with the DNA duplex distal to the site of damage. An unusual aspect of the EndoIII–DNA complex is the extensive number of contacts the enzyme makes with the strand of DNA complementary to the one containing the lesion. The structure also provided an atomic basis for understanding the preference of EndoIII to repair lesions paired opposite guanine residues in DNA; the enzyme uses mainchain carbonyl oxygens to recognize the Watson–Crick face of the guanine opposite the lesion. A unique and interesting feature of MutY is that it removes a normal adenine base when paired opposite the mutagenic lesion oxoG. The structural basis of the recognition of a lesion outside of the MutY active site www.sciencedirect.com
DNA glycosylases Fromme, Banerjee and Verdine 47
Figure 4
C-terminal domain
Catalytic domain
oxoG
N
Adenine C Current Opinion in Structural Biology
Structure of MutY bound to DNA containing an AoxoG pair (PDB code 1RRQ [39]). DNA is shown in gold CPK format, with the oxoG base colored red and the adenine base colored purple. MutY is shown in ribbon format with termini labeled. The C-terminal domain, on the left, is responsible for oxoG recognition. The [4Fe–4S] cluster and a calcium ion are shown as spheres.
has been highly anticipated. The structure of MutY in complex with DNA containing an AoxoG mispair (Figure 4) was recently determined [39] with the aid of disulfide cross-linking [40]. The enzyme recognizes oxoG using hydrogen bonds by sandwiching the oxoGcontaining strand of DNA between the N- and C-terminal domains. The oxoG residue remains intrahelical, but is bound in the anti glycosidic conformation, in contrast to the syn conformation found when paired with adenine in DNA. This observation impacts hypotheses concerning both the search mechanism and the binding events preceding catalysis. The structure also provides a clear explanation for the deleterious effects of two MutY polymorphisms found in some hereditary colon cancers, as these mutations interfere with the faithful recognition of oxoG.
UDG family UDG (or Ung) is probably the most widely studied DNA glycosylase and the structural biology of repair by UDG (and the homologous enzyme Mug) is well developed [41–44]. Work on the structural biology and monofunctional mechanism of UDG continues to bear valuable fruit. Structural studies of UDG bound to an inhibitor [45], together with biochemical studies [46], suggest that electrostatic interactions and geometric strain play large roles in catalysis. Additional evidence has demonstrated the electrostatic contribution of phosphates in the DNA backbone to catalysis [47,48] and indicates that base excision occurs through a dissociative SN1-type mechanism [48,49]. Using the structure as a guide, a mutant form of UDG was engineered, via the introduction of two amino acid substitutions, to exhibit alternative specificity for cytosine when paired opposite bulky lesions [50]. www.sciencedirect.com
The determination of the crystal structure of a SMUG1– DNA complex [51] (Figure 2c) established conclusively that this protein is indeed a member of the UDG superfamily, despite having less than 10% sequence identity with any other known member. The structures of several different SMUG1–DNA complexes were determined, representing product and nonspecific DNA complexes. The mechanism of the exclusion of thymine as a substrate is proposed to be based on the presence of an ordered water molecule in the base-recognition pocket, and the interaction of the enzyme with DNA appears to be more invasive into the duplex than seen with UDG. Further studies on trapped complexes will be necessary to test this notion.
Conclusions The structural biology of DNA glycosylases has progressed rapidly in the past few years. With attention centering on complexes between DNA glycosylases and DNA, the details of lesion recognition and catalysis are coming into focus. However, many interesting questions remain to be answered. For example, despite work on MutM that has yielded structures of three distinct stages of catalysis, the catalytic mechanism remains poorly understood. Future studies will continue to bring to light the structural basis of recognition and catalysis, but the next frontier in DNA glycosylase biology concerns the nature of the damage search process. How DNA glycosylases locate damaged bases present at very low levels in the genome has remained a mystery. We are hopeful that structures of DNA glycosylases bound to undamaged DNA will begin to flush out certain aspects of the search mechanism.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Thayer MM, Ahern H, Xing D, Cunningham RP, Tainer JA: Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure. EMBO J 1995, 14:4108-4120.
3.
Nash HM, Bruner SD, Scharer OD, Kawate T, Addona TA, Spooner E, Lane WS, Verdine GL: Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily. Curr Biol 1996, 6:968-980.
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Fromme JC, Verdine GL: Structural insights into lesion recognition and repair by the bacterial 8-oxoguanine DNA glycosylase MutM. Nat Struct Biol 2002, 9:544-552. Current Opinion in Structural Biology 2004, 14:43–49
48 Protein–nucleic acid interactions
Crystal structures of MutM–DNA complexes provide snapshots of a catalytic intermediate and the product, as well as insights into recognition of the base paired opposite the lesion.
23. Norman DPG, Chung SJ, Verdine GL: Structural and biochemical exploration of a critical amino acid in human 8-oxoguanine glycosylase. Biochemistry 2003, 42:1564-1572.
8. Fromme JC, Verdine GL: DNA lesion recognition by the bacterial repair enzyme MutM. J Biol Chem 2003, 278:51543-51548. This paper reports the crystal structures of two different MutM–DNA recognition complexes. A flexible loop is seen to be mainly responsible for lesion base recognition and the basis of recognition of more than one type of lesion is revealed.
24. Drohat AC, Kwon K, Krosky DJ, Stivers JT: 3-methyladenine DNA glycosylase I is an unexpected helix-hairpin-helix superfamily member. Nat Struct Biol 2002, 9:659-664. The solution structure of 3-methyladenine glycosylase I unexpectedly revealed it to be a member of the HhH-GPD superfamily. Interestingly, the enzyme lacks the conserved catalytic aspartate, so the basis of catalysis is mysterious.
9.
Gilboa R, Zharkov DO, Golan G, Fernandes AS, Gerchman SE, Matz E, Kycia JH, Grollman AP, Shoham G: Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA. J Biol Chem 2002, 277:19811-19816.
10. Serre L, Pereira de Jesus K, Boiteux S, Zelwer C, Castaing B: Crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing DNA. EMBO J 2002, 21:2854-2865. 11. Zharkov DO, Shoham G, Grollman AP: Structural characterization of the Fpg family of DNA glycosylases. DNA Repair 2003, 2:839-862. 12. Zharkov DO, Golan G, Gilboa R, Fernandes AS, Gerchman SE, Kycia JH, Rieger RA, Grollman AP, Shoham G: Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate. EMBO J 2002, 21:789-800. The 1.25 A˚ crystal structure of a Nei–DNA covalent intermediate was the first determined of any MutM/Fpg family member in complex with DNA. 13. Sugahara M, Mikawa T, Kumasaka T, Yamamoto M, Kato R, Fukuyama K, Inoue Y, Kuramitsu S: Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile, Thermus thermophilus HB8. EMBO J 2000, 19:3857-3869. 14. Bruner SD, Norman DP, Verdine GL: Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA. Nature 2000, 403:859-866. 15. Bandaru V, Sunkara S, Wallace SS, Bond JP: A novel human DNA glycosylase that removes oxidative DNA damage and is homologous to Escherichia coli endonuclease VIII. DNA Repair 2002, 1:517-529. 16. Morland I, Rolseth V, Luna L, Rognes T, Bjoras M, Seeberg E: Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA. Nucleic Acids Res 2002, 30:4926-4936. 17. Takao M, Kanno S, Kobayashi K, Zhang QM, Yonei S, van der Horst GT, Yasui A: A back-up glycosylase in Nth1 knock-out mice is a functional Nei (endonuclease VIII) homologue. J Biol Chem 2002, 277:42205-42213. 18. Hazra TK, Izumi T, Kow YW, Mitra S: The discovery of a new family of mammalian enzymes for repair of oxidatively damaged DNA, and its physiological implications. Carcinogenesis 2003, 24:155-157. 19. Ohtsubo T, Matsuda O, Iba K, Terashima I, Sekiguchi M, Nakabeppu Y: Molecular cloning of AtMMH, an Arabidopsis thaliana ortholog of the Escherichia coli mutM gene, and analysis of functional domains of its product. Mol Gen Genet 1998, 259:577-590. 20. Hollis T, Ichikawa Y, Ellenberger T: DNA bending and a flip-out mechanism for base excision by the helix- hairpin-helix DNA glycosylase, Escherichia coli AlkA. EMBO J 2000, 19:758-766. 21. Bjoras M, Seeberg E, Luna L, Pearl LH, Barrett TE: Reciprocal ‘flipping’ underlies substrate recognition and catalytic activation by the human 8-oxo-guanine DNA glycosylase. J Mol Biol 2002, 317:171-177. 22. Fromme JC, Bruner SD, Yang W, Karplus M, Verdine GL: Product-assisted catalysis in base-excision DNA repair. Nat Struct Biol 2003, 10:204-211. Structural, biochemical and computational studies demonstrated that the catalytic mechanism of hOGG1 uses product-assisted catalysis, the only known example of this phenomenon in enzyme-catalyzed reactions. Current Opinion in Structural Biology 2004, 14:43–49
25. Kwon K, Cao C, Stivers JT: A novel zinc snap motif conveys structural stability to 3-methyladenine DNA glycosylase I. J Biol Chem 2003, 278:19442-19446. 26. Cao C, Kwon K, Jiang YL, Drohat AC, Stivers JT: Solution structure and base perturbation studies reveal a novel mode of alkylated base recognition by 3-methyladenine DNA glycosylase I. J Biol Chem 2003, 278:48012-48020. Fluorescence binding assays and the solution structure of base analogs bound to 3-methyladenine glycosylase I suggest a novel mode of lesion recognition. 27. Eichman BF, O’Rourke EJ, Radicella JP, Ellenberger T: Crystal structures of 3-methyladenine DNA glycosylase MagIII and the recognition of alkylated bases. EMBO J 2003, 22:4898-4909. The crystal structure of 3-methyladenine glycosylase III bound to two different lesion bases reveals the basis of substrate selectivity. 28. Sved J, Bird A: The expected equilibrium of the CpG dinucleotide in vertebrate genomes under a mutation model. Proc Natl Acad Sci USA 1990, 87:4692-4696. 29. Duncan BK, Miller JH: Mutagenic deamination of cytosine residues in DNA. Nature 1980, 287:560-561. 30. Hendrich B, Hardeland U, Ng HH, Jiricny J, Bird A: The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites. Nature 1999, 401:301-304. 31. Wu P, Qiu C, Sohail A, Zhang X, Bhagwat AS, Cheng X: Mismatch repair in methylated DNA. Structure and activity of the mismatch-specific thymine glycosylase domain of methyl-CpG-binding protein MBD4. J Biol Chem 2003, 278:5285-5291. In this study, the authors confirm that the isolated C-terminal domain of MBD4 is active towards TG mismatches in a CpG sequence context. The crystal structure provides a framework for modeling substrate recognition. 32. Horst JP, Fritz HJ: Counteracting the mutagenic effect of hydrolytic deamination of DNA 5-methylcytosine residues at high temperature: DNA mismatch N-glycosylase Mig.Mth of the thermophilic archaeon Methanobacterium thermoautotrophicum THF. EMBO J 1996, 15:5459-5469. 33. Mol CD, Arvai AS, Begley TJ, Cunningham RP, Tainer JA: Structure and activity of a thermostable thymine-DNA glycosylase: evidence for base twisting to remove mismatched normal DNA bases. J Mol Biol 2002, 315:373-384. 34. Volk DE, House PG, Thiviyanathan V, Luxon BA, Zhang S, Lloyd RS, Gorenstein DG: Structural similarities between MutT and the C-terminal domain of MutY. Biochemistry 2000, 39:7331-7336. 35. Abeygunawardana C, Weber DJ, Gittis AG, Frick DN, Lin J, Miller AF, Bessman MJ, Mildvan AS: Solution structure of the MutT enzyme, a nucleoside triphosphate pyrophosphohydrolase. Biochemistry 1995, 34:14997-15005. 36. Fu W, O’Handley S, Cunningham RP, Johnson MK: The role of the iron-sulfur cluster in Escherichia coli endonuclease III. A resonance Raman study. J Biol Chem 1992, 267:16135-16137. 37. Boon EM, Livingston AL, Chmiel NH, David SS, Barton JK: DNA-mediated charge transport for DNA repair. Proc Natl Acad Sci USA 2003, 100:12543-12547. 38. Fromme JC, Verdine GL: Structure of a trapped endonuclease III-DNA covalent intermediate. EMBO J 2003, 22:3461-3471. 39. Fromme JC, Banerjee A, Huang SJ, Verdine GL: Structural basis for removal of A mispaired with 8-oxoguanine by MutY. Nature 2004, in press. www.sciencedirect.com
DNA glycosylases Fromme, Banerjee and Verdine 49
The crystal structure of MutY bound to AoxoG-containing DNA was obtained with the use of disulfide cross-linking. The structure reveals an intrahelical oxoG recognition mode in which the oxoG base has rotated about its glycosidic bond.
along the reaction coordinate of uracil DNA glycosylase. Biochemistry 2003, 42:12455-12460.
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