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DNA Glycosylases: Mechanisms
DNA Glycosylases: Mechanisms Daniel J. Krosky and James T. Stivers The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
The highly accurate replication of an organism’s DNA is necessary for it to maintain genetic stability over many generations. Thus, cells need to aggressively repair DNA damage in order to prevent mutations and to eliminate toxic base modifications that can interfere with DNA replication. To this end, cells have evolved several DNA repair pathways geared toward processing different types of DNA lesions. One of these systems, the base excision repair (BER) pathway, recognizes and removes chemically modified DNA bases and replaces them with the correct nucleotides. The first step in BER is catalyzed by a family of functionally related enzymes known as DNA glycosylases that hydrolytically cleave the glycosidic bond between the damaged base and its deoxyribose sugar.
The Biological Function of DNA Glycosylases DNA bases can be damaged by a variety of mechanisms that include alkylation, deamination, oxidation, and ultraviolet light, each of which produces a different base alteration. Repair of each of these diverse lesions begins with the action of a unique DNA glycosylase (to date, eight have been identified in humans) that is specific for the particular damaged base. This hydrolysis reaction results in a common intermediate, an abasic site, which can be processed by either short- or long-patch repair. In short-patch repair, only the damaged nucleotide is replaced, whereas in long-patch repair, the damaged base and three or four additional nucleotides are excised and replaced.
appears. Examples of these include the bacterial mismatch-specific UDG (MUG), which excises uracil paired with guanine but not with adenine, and the mammalian 8-oxoguanine glycosylase (OGG), which only cleaves the oxidized base 8-oxoguanine when it is opposite cytosine. In contrast, other DNA glycosylases cleave a relatively wide spectrum of chemically related damaged bases. For instance, Escherichia coli 3-methyladenine glycosylase II (AlkA) catalyzes the hydrolysis of a wide range of cationic N-alkylated purines, such as 3-methyladenine and 7-methylguanine. In addition, AlkA can cleave undamaged purine bases, although with much lower efficiency than N-alkylated purines. Other broadspecificity glycosylases include mammalian methylpurine glycosylase (MPG), which has a substrate range similar to AlkA, and endonuclease III (Endo III), which processes a variety of oxidized pyrimidines (e.g., thymine glycol and 5-hydroxycytosine). In addition to simply excising damaged DNA bases resulting in an abasic site, a subset of bifunctional DNA glycosylases can subsequently catalyze an elimination reaction in which the 30 -phosphate of the abasic site is expelled. Members of this bifunctional DNA glycosylase family include OGG, Endo III, and pyrimidine dimer DNA glycosylase (PDG). Other enzymes in the base excision repair (BER) pathway then process the resulting repair intermediate, mostly via the shortpatch pathway.
STRUCTURAL SUPERFAMILIES DNA GLYCOSYLASES
OF
The DNA glycosylases have evolved varying degrees of substrate specificity. For example, uracil –DNA glycosylase (UDG) will efficiently cleave uracil that has been misincorporated into DNA, but not thymine, which only differs from uracil by one methyl group. Other DNA glycosylases not only possess high specificity for the cleaved base, but also for the context in which it
Even though DNA glycosylases catalyze similar reactions, these enzymes share minimal primary sequence homology with one another. However, X-ray crystallography and nuclear magnetic resonance (NMR) analyses have revealed that many of these proteins share a common fold and have allowed the classification of these proteins into several structural superfamilies. One of these structural superfamilies is defined by the helix-hairpin-helix (HhH) motif, which is found in many non-sequence-specific DNA-binding proteins. This a-helical fold allows these enzymes to make
Encyclopedia of Biological Chemistry, Volume 1. q 2004, Elsevier Inc. All Rights Reserved.
614
Types of DNA Glycosylases SUBSTRATE RANGE
AND
SPECIFICITY
DNA GLYCOSYLASES: MECHANISMS
nonspecific contacts with the DNA phosphodiester backbone, thus permitting sequence-independent excision of their cognate lesions. Other DNA glycosylases, such as UDG, have evolved alternate structural motifs that also result in nonspecific DNA binding and fufill the same function as the HhH domain.
Functional Commonalities of DNA Glycosylases BASE FLIPPING Despite differences in substrate specificity, structure, and primary sequence, all DNA glycosylases are functionally related in their ability to extrude DNA bases from the double helix and to catalyze the hydrolysis of the glycosidic bond between the cognate base and the deoxyribose. Extruding a base from duplex DNA, or base flipping, is the process by which an enzyme facilitates the rotation of the DNA phosphodiester backbone such that a base flips from within the double helix to an extrahelical position located within the binding pocket of the enzyme (Figure 1). The most common mode of base flipping occurs when the enzyme flips the damaged base from the DNA double helix. However, some DNA glycosylases use different base-flipping strategies. PDG, which is responsible for cleaving the glycosidic bond of thymine photodimers,
615
flips the adenine that is opposite the target base. E. coli MutY, which removes adenine misincorporated opposite to the oxidized base 8-oxoguanine, appears to flip both bases from the duplex. There are several elements common to the baseflipping mechanisms of DNA glycosylases. First, the enzyme makes extensive nonspecific contacts with the phosphodiester backbone of the DNA (Figure 1). In addition to allowing sequence-independent binding of the DNA glycosylase to DNA, these protein – DNA interactions provide a stable architecture from which the enzyme can distort the DNA. All structural studies of DNA glycosylases bound to DNA have revealed that the DNA is significantly bent at the site of enzyme binding, albeit to differing degrees with different enzymes (Figure 1). Although the exact role of DNA bending is unclear, it may help certain glycosylases locate lesions in DNA. In addition, computational studies have suggested that DNA bending may play an important role in lowering the activation barrier for base flipping, thus providing an explanation for why DNA bending is such a ubiquitous feature of these interactions (Figure 1). Finally, all DNA glycosylases insert a bulky amino acid side chain, such as leucine or phenylalanine, inside the DNA double helix (Figure 1). This group serves as a structural wedge to both push the base out and to act as a barrier to prevent the base from slipping back into the double helix.
FIGURE 1 Schematic representation of base flipping and other common interactions of DNA glycosylases with DNA. Bp, damaged base; (---), hydrogen bond.
616
DNA GLYCOSYLASES: MECHANISMS
Base flipping plays an important role in facilitating base recognition and glycosidic bond cleavage. Extrusion of the base juxtaposes its unique functional groups with complementary groups in the enzyme active site. These specific interactions promote the recognition of the damaged base and largely exclude binding of undamaged bases. Differences in the amino acid residues of the base binding pocket among DNA glycosylases, in part, account for differences in their substrate specificity and catalytic mechanisms (see later discussion). Base flipping also exposes the anomeric carbon (the deoxyribose carbon that is attached to the base) to attack by water in the enzyme active site. Thus, base flipping enables DNA glycosylases to couple damaged base recognition to enzyme catalysis.
site of AlkA lacks the constellation of highly specific hydrogen-bonding groups that line the active sites of DNA glycosylases that recognize neutral damaged bases. Instead, its active site is rich in aromatic amino acids, such as tryptophan and tyrosine, that allow AlkA to bind purine bases of varying shape and account for the promiscuous activity of this enzyme. The unique chemical character of the AlkA active site may facilitate damaged-base recognition by forming favorable stacking interactions between the cationic N-alkylpurine and the aromatic side chains. Alternatively, positioning the cationic damaged base in a hydrophobic active site may serve to lower the activation barrier by electrostatic destabilization of the charged ground state. These key questions are still under active investigation.
CATALYTIC STRATEGIES FOR GLYCOSIDIC BOND HYDROLYSIS
The Nature of the Transition State
At neutral pH and ambient temperature, the glycosidic bond of a deoxynucleotide is a very stable linkage toward hydrolysis (t1=2 ¼ 109 years for thymidine; t1=2 ¼ 3:9 years for deoxyadenosine). The stability of this bond stems in part from the poor leaving-group ability of the electron-rich nucleobase, the poor nucleophilicity of water, and the poor electrophilicity of the anomeric carbon. DNA glycosylases have evolved remarkably similar strategies to overcome these chemical problems and remove these lesions rapidly enough to preserve the integrity of the genomic information.
Making the Cognate Nucleobase a Better Leaving Group Most neutral deoxynucleotide bases are poor leaving groups because they cannot effectively stabilize the developing negative charge that forms on the base during the reaction. Some DNA glycosylases stabilize this negative charge by donating specific hydrogen bonds from active-site residues to acceptors on the base (Figure 1). This type of catalysis has been most clearly shown for UDG, in which an unusually strong hydrogen bond from a histidine residue in the enzyme active site stabilizes the uracil anion leaving group. In other cases, leaving group departure may be facilitated by the full transfer of a proton from an active site donor to a proton-accepting atom on the nucleobase, as has been suggested for MutY. N-alkylated purines (i.e., 3-methyladenine and 7-methylguanine) are unique in that they are positively charged and electron-deficient; therefore, they are excellent leaving groups in the absence of any enzymatic activation. Accordingly, DNA glycosylases use different types of catalytic interventions to affect the hydrolysis of these labile bases. For example, the cationic base binding
The rupture of the glycosidic bond in DNA can proceed through two limiting routes (Figure 2). In the first route (Figure 2A), the attack of water is coincident with departure of the leaving base, resulting in a concerted transition state. In a concerted reaction, the anomeric carbon is relatively electron rich, and little negative charge has built up on the leaving nucleobase. In the second route (Figure 2B), the bond between the anomeric carbon and the nucleobase is completely broken before the new bond with water forms, and the reaction proceeds via a stepwise mechanism with a discrete intermediate. Thus, in a stepwise reaction, the anomeric carbon is electron poor and has a significant positive charge, and the bonding electrons have migrated fully onto the leaving-group nucleobase. Although comprehensive mechanistic studies for most DNA glycosylases have not been performed, it appears that these enzymes may catalyze their reactions using mechanisms in which the glycosidic bond is mostly broken and the new bond between water and the deoxyribose is only partially formed. In support of these electronic features, custom oligonucleotides containing a positively charged deoxyribose analogue that mimics the electron-deficient anomeric carbon at the transition state are potent inhibitors of several DNA glycosylases, including UDG and AlkA (Figure 2C). Such inhibitors of DNA glycosylases may someday find use in increasing the efficacy of anticancer agents that modify the bases of DNA by disabling the repair pathways that reverse the effects of these chemotherapeutics. One way in which DNA glycosylases stabilize stepwise reactions is by surrounding the positively charged deoxyribose with negatively charged groups. For example, UDG engulfs the cationic deoxyribose intermediate with a negatively charged aspartate residue, DNA phosphodiester groups, and the uracil anion leaving group.
DNA GLYCOSYLASES: MECHANISMS
617
FIGURE 2 Two limiting reaction pathways for DNA glycosylases. (A) Concerted pathway; (B) stepwise pathway; (C) DNA glycosylase inhibitor based on the cationic deoxyribose intermediate of the stepwise reaction.
Helping Water Attack the Anomeric Carbon Most DNA glycosylases possess a polar amino acid near the anomeric carbon of the target deoxyribose that can accept a hydrogen bond or proton from the attacking water (i.e., aspartate, glutamate, or asparagine) (Figure 1). Depending on the mechanism, this group may simply orient the water close to the anomeric carbon or, alternatively, may actively facilitate its attack by deprotonation. In concerted reactions, when there is significant bond formation between the attacking water and the anomeric carbon in the transition state, the activation barrier is lessened by partial or full deprotonation of the water. For stepwise reactions, the water needs only to be oriented close to the positively charged anomeric carbon because such species are highly reactive even with poor nucleophiles such as water (Figure 2B).
SEE ALSO
THE
FOLLOWING ARTICLE
DNA Base Excision Repair
between the reactants and the transition state (the activation barrier) determines the rate of reaction.
FURTHER READING Hollis, T., Ichikawa, Y., and Ellenberger, T. (2000). DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycoyslase, Escherichia coli AlkA. EMBO J. 19, 758–766. Jiang, Y. L., Drohat, A. C., Ichikawa, Y., and Stivers, J. T. (2002). Probing the limits of electrostatic catalysis by uracil DNA glycosylase using transition state mimicry and mutagenesis. J. Biol. Chem. 277, 15385–15392. Pearl, L. H. (2000). Structure and function in the uracil– DNA glycosylase superfamily. Mutat. Res. 460, 165–181. Stivers, J. T., and Drohat, A. C. (2001). Uracil DNA glycosylase: Insights from a master catalyst. Arch. Biochem. Biophys. 396, 1 –9. Stivers, J. T., and Jiang, Y. L. (2003). A mechanistic perspective on the chemistry of DNA repair glycosylases. Chem. Rev. 103, 2729–2759.
BIOGRAPHY
GLOSSARY
Daniel J. Krosky is currently a graduate student at the Johns Hopkins University School of Medicine, studying the energetics of base flipping by uracil DNA glycosylase. Prior to attending Johns Hopkins University, he was a Senior Research Associate at AstraZeneca R&D, Boston, developing novel antibacterial agents.
abasic site A deoxyribose residue in DNA that lacks a base. base flipping The process by which an enzyme extrudes a base from within the DNA double helix into an extrahelical position inside the active site of the enzyme. DNA glycosylase A family of functionally related enzymes that catalyze the hydrolytic cleavage of a damaged base from DNA. glycosidic bond The bond connecting the anomeric carbon of the deoxyribose to the nitrogen on the purine or pyrimidine base. transition state The highest potential energy species in the overall transformation of reactants to products. The difference in energy
James T. Stivers is an Associate Professor in the Department of Pharmacology and Molecular Sciences at Johns Hopkins Medical School. His research focuses on understanding the nature of enzyme catalysis and inhibition for a number of enzymes involved in DNA repair and recombination. He obtained his Ph.D. in biochemistry from Johns Hopkins University in 1992. He received postdoctoral training in heteronuclear NMR and enzymology in the laboratory of Professor Albert Mildvan at Johns Hopkins Medical School.
The highly accurate replication of an organism’s DNA is necessary for it to maintain genetic stability over many generations. Thus, cells need to aggressively repair DNA damage in order to prevent mutations and to eliminate toxic base modifications that can interfere with DNA replication. To this end, cells have evolved several DNA repair pathways geared toward processing different types of DNA lesions. One of these systems, the base excision repair (BER) pathway, recognizes and removes chemically modified DNA bases and replaces them with the correct nucleotides. The first step in BER is catalyzed by a family of functionally related enzymes known as DNA glycosylases that hydrolytically cleave the glycosidic bond between the damaged base and its deoxyribose sugar.
The Biological Function of DNA Glycosylases DNA bases can be damaged by a variety of mechanisms that include alkylation, deamination, oxidation, and ultraviolet light, each of which produces a different base alteration. Repair of each of these diverse lesions begins with the action of a unique DNA glycosylase (to date, eight have been identified in humans) that is specific for the particular damaged base. This hydrolysis reaction results in a common intermediate, an abasic site, which can be processed by either short- or long-patch repair. In short-patch repair, only the damaged nucleotide is replaced, whereas in long-patch repair, the damaged base and three or four additional nucleotides are excised and replaced.
appears. Examples of these include the bacterial mismatch-specific UDG (MUG), which excises uracil paired with guanine but not with adenine, and the mammalian 8-oxoguanine glycosylase (OGG), which only cleaves the oxidized base 8-oxoguanine when it is opposite cytosine. In contrast, other DNA glycosylases cleave a relatively wide spectrum of chemically related damaged bases. For instance, Escherichia coli 3-methyladenine glycosylase II (AlkA) catalyzes the hydrolysis of a wide range of cationic N-alkylated purines, such as 3-methyladenine and 7-methylguanine. In addition, AlkA can cleave undamaged purine bases, although with much lower efficiency than N-alkylated purines. Other broadspecificity glycosylases include mammalian methylpurine glycosylase (MPG), which has a substrate range similar to AlkA, and endonuclease III (Endo III), which processes a variety of oxidized pyrimidines (e.g., thymine glycol and 5-hydroxycytosine). In addition to simply excising damaged DNA bases resulting in an abasic site, a subset of bifunctional DNA glycosylases can subsequently catalyze an elimination reaction in which the 30 -phosphate of the abasic site is expelled. Members of this bifunctional DNA glycosylase family include OGG, Endo III, and pyrimidine dimer DNA glycosylase (PDG). Other enzymes in the base excision repair (BER) pathway then process the resulting repair intermediate, mostly via the shortpatch pathway.
STRUCTURAL SUPERFAMILIES DNA GLYCOSYLASES
OF
The DNA glycosylases have evolved varying degrees of substrate specificity. For example, uracil –DNA glycosylase (UDG) will efficiently cleave uracil that has been misincorporated into DNA, but not thymine, which only differs from uracil by one methyl group. Other DNA glycosylases not only possess high specificity for the cleaved base, but also for the context in which it
Even though DNA glycosylases catalyze similar reactions, these enzymes share minimal primary sequence homology with one another. However, X-ray crystallography and nuclear magnetic resonance (NMR) analyses have revealed that many of these proteins share a common fold and have allowed the classification of these proteins into several structural superfamilies. One of these structural superfamilies is defined by the helix-hairpin-helix (HhH) motif, which is found in many non-sequence-specific DNA-binding proteins. This a-helical fold allows these enzymes to make
Encyclopedia of Biological Chemistry, Volume 1. q 2004, Elsevier Inc. All Rights Reserved.
614
Types of DNA Glycosylases SUBSTRATE RANGE
AND
SPECIFICITY
DNA GLYCOSYLASES: MECHANISMS
nonspecific contacts with the DNA phosphodiester backbone, thus permitting sequence-independent excision of their cognate lesions. Other DNA glycosylases, such as UDG, have evolved alternate structural motifs that also result in nonspecific DNA binding and fufill the same function as the HhH domain.
Functional Commonalities of DNA Glycosylases BASE FLIPPING Despite differences in substrate specificity, structure, and primary sequence, all DNA glycosylases are functionally related in their ability to extrude DNA bases from the double helix and to catalyze the hydrolysis of the glycosidic bond between the cognate base and the deoxyribose. Extruding a base from duplex DNA, or base flipping, is the process by which an enzyme facilitates the rotation of the DNA phosphodiester backbone such that a base flips from within the double helix to an extrahelical position located within the binding pocket of the enzyme (Figure 1). The most common mode of base flipping occurs when the enzyme flips the damaged base from the DNA double helix. However, some DNA glycosylases use different base-flipping strategies. PDG, which is responsible for cleaving the glycosidic bond of thymine photodimers,
615
flips the adenine that is opposite the target base. E. coli MutY, which removes adenine misincorporated opposite to the oxidized base 8-oxoguanine, appears to flip both bases from the duplex. There are several elements common to the baseflipping mechanisms of DNA glycosylases. First, the enzyme makes extensive nonspecific contacts with the phosphodiester backbone of the DNA (Figure 1). In addition to allowing sequence-independent binding of the DNA glycosylase to DNA, these protein – DNA interactions provide a stable architecture from which the enzyme can distort the DNA. All structural studies of DNA glycosylases bound to DNA have revealed that the DNA is significantly bent at the site of enzyme binding, albeit to differing degrees with different enzymes (Figure 1). Although the exact role of DNA bending is unclear, it may help certain glycosylases locate lesions in DNA. In addition, computational studies have suggested that DNA bending may play an important role in lowering the activation barrier for base flipping, thus providing an explanation for why DNA bending is such a ubiquitous feature of these interactions (Figure 1). Finally, all DNA glycosylases insert a bulky amino acid side chain, such as leucine or phenylalanine, inside the DNA double helix (Figure 1). This group serves as a structural wedge to both push the base out and to act as a barrier to prevent the base from slipping back into the double helix.
FIGURE 1 Schematic representation of base flipping and other common interactions of DNA glycosylases with DNA. Bp, damaged base; (---), hydrogen bond.
616
DNA GLYCOSYLASES: MECHANISMS
Base flipping plays an important role in facilitating base recognition and glycosidic bond cleavage. Extrusion of the base juxtaposes its unique functional groups with complementary groups in the enzyme active site. These specific interactions promote the recognition of the damaged base and largely exclude binding of undamaged bases. Differences in the amino acid residues of the base binding pocket among DNA glycosylases, in part, account for differences in their substrate specificity and catalytic mechanisms (see later discussion). Base flipping also exposes the anomeric carbon (the deoxyribose carbon that is attached to the base) to attack by water in the enzyme active site. Thus, base flipping enables DNA glycosylases to couple damaged base recognition to enzyme catalysis.
site of AlkA lacks the constellation of highly specific hydrogen-bonding groups that line the active sites of DNA glycosylases that recognize neutral damaged bases. Instead, its active site is rich in aromatic amino acids, such as tryptophan and tyrosine, that allow AlkA to bind purine bases of varying shape and account for the promiscuous activity of this enzyme. The unique chemical character of the AlkA active site may facilitate damaged-base recognition by forming favorable stacking interactions between the cationic N-alkylpurine and the aromatic side chains. Alternatively, positioning the cationic damaged base in a hydrophobic active site may serve to lower the activation barrier by electrostatic destabilization of the charged ground state. These key questions are still under active investigation.
CATALYTIC STRATEGIES FOR GLYCOSIDIC BOND HYDROLYSIS
The Nature of the Transition State
At neutral pH and ambient temperature, the glycosidic bond of a deoxynucleotide is a very stable linkage toward hydrolysis (t1=2 ¼ 109 years for thymidine; t1=2 ¼ 3:9 years for deoxyadenosine). The stability of this bond stems in part from the poor leaving-group ability of the electron-rich nucleobase, the poor nucleophilicity of water, and the poor electrophilicity of the anomeric carbon. DNA glycosylases have evolved remarkably similar strategies to overcome these chemical problems and remove these lesions rapidly enough to preserve the integrity of the genomic information.
Making the Cognate Nucleobase a Better Leaving Group Most neutral deoxynucleotide bases are poor leaving groups because they cannot effectively stabilize the developing negative charge that forms on the base during the reaction. Some DNA glycosylases stabilize this negative charge by donating specific hydrogen bonds from active-site residues to acceptors on the base (Figure 1). This type of catalysis has been most clearly shown for UDG, in which an unusually strong hydrogen bond from a histidine residue in the enzyme active site stabilizes the uracil anion leaving group. In other cases, leaving group departure may be facilitated by the full transfer of a proton from an active site donor to a proton-accepting atom on the nucleobase, as has been suggested for MutY. N-alkylated purines (i.e., 3-methyladenine and 7-methylguanine) are unique in that they are positively charged and electron-deficient; therefore, they are excellent leaving groups in the absence of any enzymatic activation. Accordingly, DNA glycosylases use different types of catalytic interventions to affect the hydrolysis of these labile bases. For example, the cationic base binding
The rupture of the glycosidic bond in DNA can proceed through two limiting routes (Figure 2). In the first route (Figure 2A), the attack of water is coincident with departure of the leaving base, resulting in a concerted transition state. In a concerted reaction, the anomeric carbon is relatively electron rich, and little negative charge has built up on the leaving nucleobase. In the second route (Figure 2B), the bond between the anomeric carbon and the nucleobase is completely broken before the new bond with water forms, and the reaction proceeds via a stepwise mechanism with a discrete intermediate. Thus, in a stepwise reaction, the anomeric carbon is electron poor and has a significant positive charge, and the bonding electrons have migrated fully onto the leaving-group nucleobase. Although comprehensive mechanistic studies for most DNA glycosylases have not been performed, it appears that these enzymes may catalyze their reactions using mechanisms in which the glycosidic bond is mostly broken and the new bond between water and the deoxyribose is only partially formed. In support of these electronic features, custom oligonucleotides containing a positively charged deoxyribose analogue that mimics the electron-deficient anomeric carbon at the transition state are potent inhibitors of several DNA glycosylases, including UDG and AlkA (Figure 2C). Such inhibitors of DNA glycosylases may someday find use in increasing the efficacy of anticancer agents that modify the bases of DNA by disabling the repair pathways that reverse the effects of these chemotherapeutics. One way in which DNA glycosylases stabilize stepwise reactions is by surrounding the positively charged deoxyribose with negatively charged groups. For example, UDG engulfs the cationic deoxyribose intermediate with a negatively charged aspartate residue, DNA phosphodiester groups, and the uracil anion leaving group.
DNA GLYCOSYLASES: MECHANISMS
617
FIGURE 2 Two limiting reaction pathways for DNA glycosylases. (A) Concerted pathway; (B) stepwise pathway; (C) DNA glycosylase inhibitor based on the cationic deoxyribose intermediate of the stepwise reaction.
Helping Water Attack the Anomeric Carbon Most DNA glycosylases possess a polar amino acid near the anomeric carbon of the target deoxyribose that can accept a hydrogen bond or proton from the attacking water (i.e., aspartate, glutamate, or asparagine) (Figure 1). Depending on the mechanism, this group may simply orient the water close to the anomeric carbon or, alternatively, may actively facilitate its attack by deprotonation. In concerted reactions, when there is significant bond formation between the attacking water and the anomeric carbon in the transition state, the activation barrier is lessened by partial or full deprotonation of the water. For stepwise reactions, the water needs only to be oriented close to the positively charged anomeric carbon because such species are highly reactive even with poor nucleophiles such as water (Figure 2B).
SEE ALSO
THE
FOLLOWING ARTICLE
DNA Base Excision Repair
between the reactants and the transition state (the activation barrier) determines the rate of reaction.
FURTHER READING Hollis, T., Ichikawa, Y., and Ellenberger, T. (2000). DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycoyslase, Escherichia coli AlkA. EMBO J. 19, 758–766. Jiang, Y. L., Drohat, A. C., Ichikawa, Y., and Stivers, J. T. (2002). Probing the limits of electrostatic catalysis by uracil DNA glycosylase using transition state mimicry and mutagenesis. J. Biol. Chem. 277, 15385–15392. Pearl, L. H. (2000). Structure and function in the uracil– DNA glycosylase superfamily. Mutat. Res. 460, 165–181. Stivers, J. T., and Drohat, A. C. (2001). Uracil DNA glycosylase: Insights from a master catalyst. Arch. Biochem. Biophys. 396, 1 –9. Stivers, J. T., and Jiang, Y. L. (2003). A mechanistic perspective on the chemistry of DNA repair glycosylases. Chem. Rev. 103, 2729–2759.
BIOGRAPHY
GLOSSARY
Daniel J. Krosky is currently a graduate student at the Johns Hopkins University School of Medicine, studying the energetics of base flipping by uracil DNA glycosylase. Prior to attending Johns Hopkins University, he was a Senior Research Associate at AstraZeneca R&D, Boston, developing novel antibacterial agents.
abasic site A deoxyribose residue in DNA that lacks a base. base flipping The process by which an enzyme extrudes a base from within the DNA double helix into an extrahelical position inside the active site of the enzyme. DNA glycosylase A family of functionally related enzymes that catalyze the hydrolytic cleavage of a damaged base from DNA. glycosidic bond The bond connecting the anomeric carbon of the deoxyribose to the nitrogen on the purine or pyrimidine base. transition state The highest potential energy species in the overall transformation of reactants to products. The difference in energy
James T. Stivers is an Associate Professor in the Department of Pharmacology and Molecular Sciences at Johns Hopkins Medical School. His research focuses on understanding the nature of enzyme catalysis and inhibition for a number of enzymes involved in DNA repair and recombination. He obtained his Ph.D. in biochemistry from Johns Hopkins University in 1992. He received postdoctoral training in heteronuclear NMR and enzymology in the laboratory of Professor Albert Mildvan at Johns Hopkins Medical School.