FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2738334611> ?p ?o ?g. }

• W2738334611 endingPage "16080" @default.
• W2738334611 startingPage "16070" @default.
• W2738334611 abstract "DNA repair enzymes recognize and remove damaged bases that are embedded in the duplex. To gain access, most enzymes use nucleotide flipping, whereby the target nucleotide is rotated 180° into the active site. In human alkyladenine DNA glycosylase (AAG), the enzyme that initiates base excision repair of alkylated bases, the flipped-out nucleotide is stabilized by intercalation of the side chain of tyrosine 162 that replaces the lesion nucleobase. Previous kinetic studies provided evidence for the formation of a transient complex that precedes the stable flipped-out complex, but it is not clear how this complex differs from nonspecific complexes. We used site-directed mutagenesis and transient-kinetic approaches to investigate the timing of Tyr162 intercalation for AAG. The tryptophan substitution (Y162W) appeared to be conservative, because the mutant protein retained a highly favorable equilibrium constant for flipping the 1,N6-ethenoadenine (ϵA) lesion, and the rate of N-glycosidic bond cleavage was identical to that of the wild-type enzyme. We assigned the tryptophan fluorescence signal from Y162W by removing two native tryptophan residues (W270A/W284A). Stopped-flow experiments then demonstrated that the change in tryptophan fluorescence of the Y162W mutant is extremely rapid upon binding to either damaged or undamaged DNA, much faster than the lesion-recognition and nucleotide flipping steps that were independently determined by monitoring the ϵA fluorescence. These observations suggest that intercalation by this aromatic residue is one of the earliest steps in the search for DNA damage and that this interaction is important for the progression of AAG from nonspecific searching to specific-recognition complexes. DNA repair enzymes recognize and remove damaged bases that are embedded in the duplex. To gain access, most enzymes use nucleotide flipping, whereby the target nucleotide is rotated 180° into the active site. In human alkyladenine DNA glycosylase (AAG), the enzyme that initiates base excision repair of alkylated bases, the flipped-out nucleotide is stabilized by intercalation of the side chain of tyrosine 162 that replaces the lesion nucleobase. Previous kinetic studies provided evidence for the formation of a transient complex that precedes the stable flipped-out complex, but it is not clear how this complex differs from nonspecific complexes. We used site-directed mutagenesis and transient-kinetic approaches to investigate the timing of Tyr162 intercalation for AAG. The tryptophan substitution (Y162W) appeared to be conservative, because the mutant protein retained a highly favorable equilibrium constant for flipping the 1,N6-ethenoadenine (ϵA) lesion, and the rate of N-glycosidic bond cleavage was identical to that of the wild-type enzyme. We assigned the tryptophan fluorescence signal from Y162W by removing two native tryptophan residues (W270A/W284A). Stopped-flow experiments then demonstrated that the change in tryptophan fluorescence of the Y162W mutant is extremely rapid upon binding to either damaged or undamaged DNA, much faster than the lesion-recognition and nucleotide flipping steps that were independently determined by monitoring the ϵA fluorescence. These observations suggest that intercalation by this aromatic residue is one of the earliest steps in the search for DNA damage and that this interaction is important for the progression of AAG from nonspecific searching to specific-recognition complexes. Nucleobases of DNA readily react with intracellular and environmental agents to form damaged base lesions. Failure to repair these base lesions leads to mutations or cell death (1Lindahl T. Instability and decay of the primary structure of DNA.Nature. 1993; 362: 709-715Crossref PubMed Scopus (4325) Google Scholar, 2Robertson A.B. Klungland A. Rognes T. Leiros I. DNA repair in mammalian cells: base excision repair: the long and short of it.Cell Mol. Life Sci. 2009; 66: 981-993Crossref PubMed Scopus (428) Google Scholar). The base excision repair pathway is the main mechanism by which single base lesions in DNA are repaired (3Krokan H.E. Bjørås M. Base excision repair.Cold Spring Harb. Perspect. Biol. 2013; 5: a012583Crossref PubMed Scopus (690) Google Scholar). The base excision repair pathway is initiated by a DNA glycosylase that is responsible for finding the damaged site and catalyzing the hydrolysis of the N-glycosidic bond. Subsequent action of an abasic-site specific endonuclease, a 5′-deoxyribose phosphate lyase, a DNA repair polymerase, and a DNA ligase are required to restore the correct DNA sequence, using the intact strand as a template. There are 11 known human DNA glycosylases that belong to 4 different structural superfamilies. Despite their structural differences, all have adopted the common strategy of nucleotide flipping to access base lesions in duplex DNA. Nucleotide or base flipping describes the complete 180° rotation of a nucleotide out of the DNA duplex to position the target nucleobase into an enzyme active site, and this general mechanism has been described for many types of DNA modifying enzymes (4Roberts R.J. Cheng X. Base flipping.Annu. Rev. Biochem. 1998; 67: 181-198Crossref PubMed Scopus (307) Google Scholar). Human alkyladenine DNA glycosylase (AAG) 2The abbreviations used are: AAGalkyladenine DNA glycosylase, also known as methylpurine DNA glycosylase and 3-methyladenine DNA glycosylaseϵA1,N6-ethenoadenineHxhypoxanthineIdeoxyinosineNaMESsodium 2-(N-morpholino)ethanesulfonate. is a monomeric DNA glycosylase responsible for recognizing a wide variety of structurally diverse deaminated and alkylated purine lesions (5O'Connor T.R. Purification and characterization of human 3-methyladenine-DNA glycosylase.Nucleic Acids Res. 1993; 21: 5561-5569Crossref PubMed Scopus (135) Google Scholar6Engelward B.P. Weeda G. Wyatt M.D. Broekhof J.L. de Wit J. Donker I. Allan J.M. Gold B. Hoeijmakers J.H. Samson L.D. Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 13087-13092Crossref PubMed Scopus (205) Google Scholar, 7O'Brien P.J. Ellenberger T. Dissecting the broad substrate specificity of human 3-methyladenine-DNA glycosylase.J. Biol. Chem. 2004; 279: 9750-9757Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar8Ibeanu G. Hartenstein B. Dunn W.C. Chang L.Y. Hofmann E. Coquerelle T. Mitra S. Kaina B. Overexpression of human DNA repair protein N-methylpurine-DNA glycosylase results in the increased removal of N-methylpurines in DNA without a concomitant increase in resistance to alkylating agents in Chinese hamster ovary cells.Carcinogenesis. 1992; 13: 1989-1995Crossref PubMed Scopus (69) Google Scholar). The minimal kinetic mechanism for the recognition, flipping, and excision of 1,N6-ethenoadenine (ϵA) was previously determined by following the changes in the intrinsic fluorescence of this lesion (9Wolfe A.E. O'Brien P.J. Kinetic mechanism for the flipping and excision of 1,N6-ethenoadenine by human alkyladenine DNA glycosylase.Biochemistry. 2009; 48: 11357-11369Crossref PubMed Scopus (41) Google Scholar, 10Hendershot J.M. Wolfe A.E. O'Brien P.J. Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase.Biochemistry. 2011; 50: 1864-1874Crossref PubMed Scopus (12) Google Scholar). Transient-kinetic experiments indicated that an initial recognition complex is rapidly and reversibly formed in which the ϵA lesion is partially unstacked. Subsequently the ϵA lesion is flipped out of the duplex into the active site to form a stable specific-recognition complex that positions the N-glycosidic bond for hydrolysis. This kinetic model has been guided by the crystal structures of AAG bound specifically to DNA that implicate a highly conserved β hairpin (β3β4) in specific DNA recognition (11Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision.Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 12Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (215) Google Scholar). The tip of this β hairpin projects into the minor groove, placing the side chain of Tyr162 within the duplex where it occupies the space vacated by the base lesion (Fig. 1). alkyladenine DNA glycosylase, also known as methylpurine DNA glycosylase and 3-methyladenine DNA glycosylase 1,N6-ethenoadenine hypoxanthine deoxyinosine sodium 2-(N-morpholino)ethanesulfonate. Recent characterization of a mutant enzyme lacking this tyrosine side chain (Y162A) demonstrated that this mutation destabilizes the flipped-out specific-recognition complex because of an accelerated rate of unflipping (13Hendershot J.M. O'Brien P.J. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.Nucleic Acids Res. 2014; 42: 12681-12690Crossref PubMed Scopus (13) Google Scholar). This is consistent with the tyrosine serving as a steric plug to prevent the lesion nucleotide from returning to the DNA duplex. In addition, the Y162A mutant of AAG catalyzes nucleotide flipping 50-fold faster than observed for the WT enzyme, and the initial DNA-binding and DNA-searching steps became too fast to measure by stopped flow (13Hendershot J.M. O'Brien P.J. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.Nucleic Acids Res. 2014; 42: 12681-12690Crossref PubMed Scopus (13) Google Scholar). These observations suggested that tyrosine 162 plays roles beyond serving as a plug. Models for enzyme-catalyzed nucleotide flipping vary from transient capture of extrahelical bases to active destabilization of the duplex. These classes of models can be distinguished by the timing of DNA intercalation, because intercalation happens after nucleotide flipping in the transient capture models and happens earlier than flipping in the active destabilization models. In the current work, we investigated the timing for intercalation by tyrosine 162 in AAG-catalyzed recognition and repair of ϵA. We took the approach of mutating the intercalating residue to tryptophan (Y162W), which provides the opportunity to directly probe the changes in environment of the intercalating residue along the reaction coordinate for binding, searching, and flipping out a damaged nucleotide. To unambiguously assign the fluorescence of the introduced tryptophan, we also mutated the native tryptophan residues that are present in the catalytic domain of AAG. We fully characterized the kinetic parameters for Y162W-catalyzed excision of ϵA to compare it to the WT enzyme and then used steady-state and rapid mixing experiments to monitor the changes in tryptophan fluorescence upon binding to undamaged and damaged DNA. This work demonstrates that substitution of the highly conserved tyrosine at the tip of the intercalating β hairpin of AAG with tryptophan (Y162W) has minimal effects on the overall kinetic parameters. The rate of ϵA excision is identical for Y162W and WT AAG, and both enzymes have similar highly favorable equilibrium constants for nucleotide flipping. The tryptophan fluorescence of Y162W AAG is rapidly quenched upon binding to DNA and does not change throughout the time scale of nucleotide flipping, which is monitored independently by changes in fluorescence of ϵA. This suggests that the tip of the β hairpin intercalates early in the search for DNA damage, and it engages with the DNA throughout the process of searching for and subsequently engaging sites of DNA damage. The Y162W variant of AAG was created via site-directed mutagenesis and purified using the same protocol as used for the WT enzyme. It behaved very similarly to the WT protein throughout the purification. We first monitored steady-state binding of Y162W to ϵA-DNA to confirm stable substrate binding and calculate the amount of active enzyme. The intrinsic fluorescence of ϵA, which can be excited at 313 nm and emits at 410 nm, provides a sensitive probe for binding directly to this damaged base (9Wolfe A.E. O'Brien P.J. Kinetic mechanism for the flipping and excision of 1,N6-ethenoadenine by human alkyladenine DNA glycosylase.Biochemistry. 2009; 48: 11357-11369Crossref PubMed Scopus (41) Google Scholar). The results demonstrate that Y162W behaves very similarly to the WT enzyme in this assay, and 100% of the enzyme is active, which is similar to the value of 86% active that was determined for the WT AAG (Fig. 2A). The strong quenching of ϵA fluorescence indicates tight binding of the flipped-out ϵA lesion in the active site (9Wolfe A.E. O'Brien P.J. Kinetic mechanism for the flipping and excision of 1,N6-ethenoadenine by human alkyladenine DNA glycosylase.Biochemistry. 2009; 48: 11357-11369Crossref PubMed Scopus (41) Google Scholar). We next tested whether the Y162W mutation affected catalysis of N-glycosidic bond cleavage by performing single-turnover experiments with enzyme in excess over ϵA-DNA substrate. Under these conditions, the hydrolysis of the N-glycosidic bond is rate-limiting for WT AAG (9Wolfe A.E. O'Brien P.J. Kinetic mechanism for the flipping and excision of 1,N6-ethenoadenine by human alkyladenine DNA glycosylase.Biochemistry. 2009; 48: 11357-11369Crossref PubMed Scopus (41) Google Scholar, 13Hendershot J.M. O'Brien P.J. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.Nucleic Acids Res. 2014; 42: 12681-12690Crossref PubMed Scopus (13) Google Scholar). The Y162W mutation does not perturb the transition state for hydrolysis, because the rate constant for ϵA excision is identical within error to that of WT AAG (Fig. 2B). This suggests that the Y162W mutation may be a very conservative substitution; however, AAG binds quickly and tightly to ϵA-DNA, and effects on earlier binding steps could be masked by the rate-limiting chemistry (10Hendershot J.M. Wolfe A.E. O'Brien P.J. Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase.Biochemistry. 2011; 50: 1864-1874Crossref PubMed Scopus (12) Google Scholar). Therefore direct binding and nucleotide flipping measurements are needed to evaluate the effects of the mutation on these steps. We used the time-dependent changes in the fluorescence of ϵA to characterize the microscopic steps involving the binding and flipping of this lesion by Y162W AAG under the same conditions previously used for the WT enzyme (13Hendershot J.M. O'Brien P.J. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.Nucleic Acids Res. 2014; 42: 12681-12690Crossref PubMed Scopus (13) Google Scholar). When a fixed concentration of ϵA-DNA duplex was mixed with increasing concentrations of Y162W, an initial decrease in fluorescence was observed that was followed by an increase in fluorescence (Fig. 3A). This was unexpected because WT AAG shows the opposite trend in binding to the ϵA-DNA substrate under the same conditions (Fig. 3A), and it raises the possibility that the tryptophan side chain of Y162W interacts directly with the ϵA lesion in the initial recognition complex prior to flipping. By analogy, tyrosine likely interacts with the ϵA lesion in the WT protein but is less effective than tryptophan at quenching the ϵA fluorescence. Although the amplitude of ϵA fluorescence change is smaller for Y162W AAG than was observed for the WT enzyme, reproducible data were obtained at different concentrations of enzyme (Fig. 3B). These traces were fit by double-exponential fits. As expected, the rate constant for the first phase (k1, obs) is linearly dependent on enzyme concentration (Fig. 3C), and this is assigned to binding and formation of the initial recognition complex (9Wolfe A.E. O'Brien P.J. Kinetic mechanism for the flipping and excision of 1,N6-ethenoadenine by human alkyladenine DNA glycosylase.Biochemistry. 2009; 48: 11357-11369Crossref PubMed Scopus (41) Google Scholar, 13Hendershot J.M. O'Brien P.J. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.Nucleic Acids Res. 2014; 42: 12681-12690Crossref PubMed Scopus (13) Google Scholar). The slope corresponds to an observed rate constant of 4 × 108 m−1 s−1, and this value is within 3-fold of the value determined for WT AAG (Table 1). The slower rate constant for the second step (k2,obs) was independent of AAG concentration and corresponds to the nucleotide flipping step with formation of the specific lesion-recognition complex (Fig. 3D). The observed rate constant for nucleotide flipping reflects an approach to equilibrium, and therefore it is equal to the sum of the rate constants for flipping and unflipping. To determine the microscopic rate constants for flipping and unflipping, it is necessary to carry out additional experiments such as pulse–chase assays that can measure the partitioning forward and backward from the flipped-out intermediate.Table 1Kinetic parameters for recognition and excision of ϵA by WT and Y162W AAGWTaThe values for WT AAG have been previously published (13).Y162Wkon (m−1s−1)(1.1 ± 0.03) × 109(0.40 ± 0.05) × 109kflip (s−1)3.6 ± 0.77.9 ± 0.7kunflip (s−1)(1.6 ± 0.3) × 10−3(5.5 ± 0.2) × 10−3KflipbThe equilibrium constant for flipping is given by the ratio of the flipping and unflipping rate constants (Kflip = kflip/kunflip).2300 ± 6001400 ± 100kmax ϵA (s−1)(8.0 ± 0.6) × 10−4(8.3 ± 0.5) × 10−4a The values for WT AAG have been previously published (13Hendershot J.M. O'Brien P.J. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.Nucleic Acids Res. 2014; 42: 12681-12690Crossref PubMed Scopus (13) Google Scholar).b The equilibrium constant for flipping is given by the ratio of the flipping and unflipping rate constants (Kflip = kflip/kunflip). Open table in a new tab We performed a pulse–chase experiment in which either WT or Y162W AAG was mixed with fluorescently labeled ϵA-DNA and then chased with an excess of pyrrolidine inhibitor DNA (Fig. 4A). Pyrrolidine is a transition state analog of AAG that binds very tightly (11Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision.Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 14Schärer O.D. Nash H.M. Jiricny J. Laval J. Verdine G.L. Specific binding of a designed pyrrolidine abasic site analog to multiple DNA glycosylases.J. Biol. Chem. 1998; 273: 8592-8597Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), making it an effective trap. The partitioning between dissociation and base excision can be measured, because the protein that dissociates is immediately bound to the inhibitor. For WT AAG, 70% dissociates from the substrate, and 30% partitions to product (Fig. 4B). This end point can be used for calculating the observed rate constant for dissociation (Equation 6). Assuming fast dissociation of AAG from nonspecific DNA, the observed rate constant is simply the rate constant for unflipping (kunflip). When the same experiment was performed for Y162W AAG, 87% of the substrate dissociated from the bound complex (Fig. 4B), indicating a 3-fold faster value for kunflip as compared with the WT enzyme (Table 1). Although the value of kunflip is slightly increased by the Y162W mutation, this rate constant remains significantly lower than the observed rate constant for flipping (k2,obs; Fig. 3D). Therefore the microscopic rate constant for flipping (kflip) is approximately equal to this observed rate constant for formation of the specific-recognition complex. The equilibrium constant for nucleotide flipping is calculated as the ratio of kflip and kunflip (Kflip = kflip/kunflip). The Y162W mutation causes only a 2-fold reduction in the Kflip value relative to the WT enzyme (Table 1), confirming that Y162W is a fairly conservative mutation. We next investigated the changes in tryptophan fluorescence for binding of Y162W AAG to DNA. AAG has multiple tryptophan residues (Trp243, Trp270, and Trp284) that could complicate the assignment of the observed tryptophan fluorescence; therefore we also sought to mutate each of these residues. Each individual tryptophan could be mutated to alanine, but the W243A mutant was poorly soluble, and the triple mutant W243A/W270A/W284A was completely insoluble (data not shown). However, the double mutant W270A/W284A was soluble and could be purified in good yield. Therefore we also introduced the Y162W mutation into this background to generate a triple mutant Y162W/W270A/W284A, which also behaved well. We confirmed that these additional mutant proteins bound tightly to ϵA-DNA and determined the concentration of active AAG as described for the Y162W mutant protein (Fig. 2A). Single-turnover excision of ϵA was found to be ∼2-fold slower for the mutant proteins with the W270A/W284A mutation, suggesting only a minor perturbation of the protein structure and ruling out large structural changes (Fig. 2B). Steady-state titrations were performed with each AAG variant, measuring the tryptophan fluorescence at increasing concentrations of ϵA-DNA (Fig. 5A). As previously reported, WT AAG exhibits 20% quenching of tryptophan fluorescence upon binding to damaged DNA (10Hendershot J.M. Wolfe A.E. O'Brien P.J. Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase.Biochemistry. 2011; 50: 1864-1874Crossref PubMed Scopus (12) Google Scholar). In contrast, Y162W AAG is quenched by 40%, suggesting that the tryptophan at position 162 is sensitive to DNA binding. The quenching of AAG fluorescence is completely eliminated by the W270A/W284A mutations, suggesting that the fluorescence of Trp243 is not sensitive to DNA binding and demonstrating a clean background for the introduction of the Y162W mutation. The fluorescence quenching of the Y162W/W270A/W284A mutant is also ∼20%, consistent with the quenching amplitudes of the WT and other mutant enzymes. The stoichiometric quenching of ϵA fluorescence by each of these AAG variants demonstrates that the specific-recognition complex was formed (Fig. 2A). To test nonspecific DNA binding, these titrations were repeated with undamaged DNA duplex, and the changes in tryptophan fluorescence are summarized in Fig. 5B. In each case, the magnitude of tryptophan quenching was almost identical whether or not the DNA contained an ϵA site. These results establish that Y162W and one or both of the pair of native tryptophan residues (Trp270/Trp284) are sensitive to nonspecific DNA binding. We next performed stopped-flow fluorescence experiments to probe the transient changes in tryptophan fluorescence that occur during the early steps associated with finding and flipping out an ϵA lesion. Y162W AAG showed a rapid quenching of fluorescence that occurred within the dead time of the stopped flow (≤2 ms) and no other detectable changes over 2 s (Fig. 6A). Under the same conditions, the ϵA fluorescence for Y162W binding to ϵA-DNA demonstrates formation of the initial recognition complex and flipping to form the specific-recognition complex (Fig. 3B). This strongly suggests that the tryptophan fluorescence is quenched upon initial binding to DNA. Consistent with this model, when the experiments were repeated with undamaged DNA, the tryptophan fluorescence was again quenched rapidly upon initial DNA binding (Fig. 6B). Because it was possible that the changes in Trp270/Trp284 were masking changes in Y162W fluorescence, the stopped-flow experiments were repeated with Y162W/W270A/W284A AAG. Once again, the tryptophan fluorescence was fully quenched upon initial DNA binding regardless of whether the DNA contained a site of damage (Fig. 6, C and D). The W270A/W284A mutant enzyme was not quenched by binding to DNA, suggesting that the introduced Y162W tryptophan in the triple mutant enzyme is responsible for the rapid quenching of tryptophan fluorescence that occurs on binding to either damaged or undamaged DNA. We did not observe any further changes in the tryptophan fluorescence throughout the entirety of the searching and flipping process. Taken together, these results suggest that tryptophan 162 intercalates rapidly into the DNA upon nonspecific DNA binding. Given the similar overall kinetic parameters of Y162W and WT AAG, we expect a similar intercalation by the native tyrosine at this position. Because AAG recognizes a wide variety of alkylated and deaminated bases, we investigated the effects of mutating Tyr162 on the maximal single-turnover rate constant for excision of Hx. We used the gel-based assay to determine glycosylase activity for the natural context for deamination of deoxyadenosine to deoxyinosine (I·T) and for a single nucleotide bulge context that is also efficiently recognized by WT AAG (15Lyons D.M. O'Brien P.J. Efficient recognition of an unpaired lesion by a DNA repair glycosylase.J. Am. Chem. Soc. 2009; 131: 17742-17743Crossref PubMed Scopus (29) Google Scholar). WT AAG removes Hx from an I·T base pair or from a single nucleotide I bulge with similar maximal rate constants (Fig. 7A). As expected, the single-turnover rate constant was independent of the concentration of enzyme under these conditions (kobs = kmax), and the rate constants are summarized in Table 2. Y162W AAG exhibits similar rate constants for excision of Hx from these same contexts, providing additional evidence that the Y162W substitution is minimally perturbing (Fig. 7B). For comparison, we revisited the previously described Y162A mutation of AAG, which greatly alters the kinetics and thermodynamics of nucleotide flipping for ϵA-DNA (13Hendershot J.M. O'Brien P.J. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.Nucleic Acids Res. 2014; 42: 12681-12690Crossref PubMed Scopus (13) Google Scholar). The Y162A mutation strongly reduced the maximal rate of Hx excision from both contexts (Fig. 7C). The 350-fold reduction in Hx excision for Y162A relative to WT AAG demonstrates the importance of an aromatic side chain for efficient engagement of the target site (Table 2).Table 2Single-turnover rate constants for excision of Hx by AAGkmax min−1krel (WT/mutant)aThe values of the relative rate constant (krel) are for I·T but similar to those calculated for the I bulge.I·TI bulgeWT2.9 ± 0.052.3 ± 0.3(1)Y162W1.2 ± 0.10.81 ± 0.132.4Y162A0.0083 ± 0.00140.0049 ± 0.0006350a The values of the relative rate constant (krel) are for I·T but similar to those calculated for the I bulge. Open table in a new tab The β3β4 hairpin and the intercalating residue, Tyr162, are highly conserved among AAG homologs, and extensive random mutagenesis failed to identify functional variants at this position (16Guo H.H. Choe J. Loeb L.A. Protein tolerance to random amino acid change.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9205-9210Crossref PubMed Scopus (226) Google Scholar). Previously this residue was shown to be critical for in vivo function, and the Y162A mutant is unable to protect cells against exogenous alkylating agents (12Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (215) Google Scholar). Biochemical studies suggest that Tyr162 plays multiple roles in the search for DNA damage. It appears to act as a plug to slow the rate of unflipping, thereby stabilizing the specific lesion-recognition complex (13Hendershot J.M. O'Brien P.J. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.Nucleic Acids Res. 2014; 42: 12681-12690Crossref PubMed Scopus (13) Google Scholar). This result is supported by crystal structures of extrahelical AAG complexes in which the side chain of Tyr162 occupies the position vacated by the flipped-out nucleobase (11Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision.Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 12Lau A.Y. Wyatt M.D. Glassner B.J. Samson L.D. Ellenberger T. Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13573-13578Crossref PubMed Scopus (215) Google Scholar, 17Lingaraju G.M. Davis C.A. Setser J.W. Samson L.D. Drennan C.L. Structural basis for the inhibition of human alkyladenine DNA glycosylase (AAG) by 3,N4-ethenocytosine-containing DNA.J. Biol. Chem. 2011; 286: 13205-13213Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In addition to this expected result, it has been shown that Tyr162 is responsible for slowing the process of nucleotide flipping (13Hendershot J.M. O'Brien P.J. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping.Nucleic Acids Res. 2014; 42: 12681-12690Crossref PubMed Scopus (13) Google Scholar). In the current work, we have characterized the kinetic parameters associated with flipping out an ϵA lesion. The kinetic parameters associated with AAG-catalyzed nucleotide flipping that were measured under identical conditions for several different Tyr162 variants, Y162A, Y162F, and Y126W, are summarized in Fig. 8. This kinetic and thermodynamic analysis establishes that both" @default.
• W2738334611 created "2017-07-31" @default.
• W2738334611 creator A5062346500 @default.
• W2738334611 creator A5086205067 @default.
• W2738334611 date "2017-09-01" @default.
• W2738334611 modified "2023-10-16" @default.
• W2738334611 title "Search for DNA damage by human alkyladenine DNA glycosylase involves early intercalation by an aromatic residue" @default.
• W2738334611 cites W1972418624 @default.
• W2738334611 cites W1972423665 @default.
• W2738334611 cites W1980349853 @default.
• W2738334611 cites W1983367240 @default.
• W2738334611 cites W1987685428 @default.
• W2738334611 cites W1987866754 @default.
• W2738334611 cites W2003447156 @default.
• W2738334611 cites W2011419587 @default.
• W2738334611 cites W2024541899 @default.
• W2738334611 cites W2034438104 @default.
• W2738334611 cites W2038898901 @default.
• W2738334611 cites W2042551931 @default.
• W2738334611 cites W2064154583 @default.
• W2738334611 cites W2067829112 @default.
• W2738334611 cites W2080985886 @default.
• W2738334611 cites W2090654092 @default.
• W2738334611 cites W2095965962 @default.
• W2738334611 cites W2099088591 @default.
• W2738334611 cites W2107043033 @default.
• W2738334611 cites W2113847441 @default.
• W2738334611 cites W2134259738 @default.
• W2738334611 cites W2154111088 @default.
• W2738334611 cites W2160419835 @default.
• W2738334611 cites W2167839384 @default.
• W2738334611 cites W4241480733 @default.
• W2738334611 doi "https://doi.org/10.1074/jbc.m117.782813" @default.
• W2738334611 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5625039" @default.
• W2738334611 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28747435" @default.
• W2738334611 hasPublicationYear "2017" @default.
• W2738334611 type Work @default.
• W2738334611 sameAs 2738334611 @default.
• W2738334611 citedByCount "5" @default.
• W2738334611 countsByYear W27383346112017 @default.
• W2738334611 countsByYear W27383346112018 @default.
• W2738334611 countsByYear W27383346112021 @default.
• W2738334611 countsByYear W27383346112022 @default.
• W2738334611 crossrefType "journal-article" @default.
• W2738334611 hasAuthorship W2738334611A5062346500 @default.
• W2738334611 hasAuthorship W2738334611A5086205067 @default.
• W2738334611 hasBestOaLocation W27383346111 @default.
• W2738334611 hasConcept C12554922 @default.
• W2738334611 hasConcept C137824038 @default.
• W2738334611 hasConcept C143425029 @default.
• W2738334611 hasConcept C178790620 @default.
• W2738334611 hasConcept C185592680 @default.
• W2738334611 hasConcept C192396546 @default.
• W2738334611 hasConcept C2778711568 @default.
• W2738334611 hasConcept C2781338088 @default.
• W2738334611 hasConcept C552990157 @default.
• W2738334611 hasConcept C55493867 @default.
• W2738334611 hasConcept C86803240 @default.
• W2738334611 hasConceptScore W2738334611C12554922 @default.
• W2738334611 hasConceptScore W2738334611C137824038 @default.
• W2738334611 hasConceptScore W2738334611C143425029 @default.
• W2738334611 hasConceptScore W2738334611C178790620 @default.
• W2738334611 hasConceptScore W2738334611C185592680 @default.
• W2738334611 hasConceptScore W2738334611C192396546 @default.
• W2738334611 hasConceptScore W2738334611C2778711568 @default.
• W2738334611 hasConceptScore W2738334611C2781338088 @default.
• W2738334611 hasConceptScore W2738334611C552990157 @default.
• W2738334611 hasConceptScore W2738334611C55493867 @default.
• W2738334611 hasConceptScore W2738334611C86803240 @default.
• W2738334611 hasFunder F4320337351 @default.
• W2738334611 hasFunder F4320337354 @default.
• W2738334611 hasIssue "39" @default.
• W2738334611 hasLocation W27383346111 @default.
• W2738334611 hasLocation W27383346112 @default.
• W2738334611 hasLocation W27383346113 @default.
• W2738334611 hasOpenAccess W2738334611 @default.
• W2738334611 hasPrimaryLocation W27383346111 @default.
• W2738334611 hasRelatedWork W2021306106 @default.
• W2738334611 hasRelatedWork W2026813999 @default.
• W2738334611 hasRelatedWork W2113261269 @default.
• W2738334611 hasRelatedWork W2131141230 @default.
• W2738334611 hasRelatedWork W2154129568 @default.
• W2738334611 hasRelatedWork W2163486875 @default.
• W2738334611 hasRelatedWork W2345318811 @default.
• W2738334611 hasRelatedWork W2738334611 @default.
• W2738334611 hasRelatedWork W2768780572 @default.
• W2738334611 hasRelatedWork W2950708332 @default.
• W2738334611 hasVolume "292" @default.
• W2738334611 isParatext "false" @default.
• W2738334611 isRetracted "false" @default.
• W2738334611 magId "2738334611" @default.
• W2738334611 workType "article" @default.