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1.
Zhou K Gao Y Hoy JA Mann FM Honzatko RB Peters RJ 《The Journal of biological chemistry》2012,287(9):6840-6850
Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg2+ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities. 相似文献
2.
Ranjana Pal Zubaidah M. Ramdzan Simran Kaur Philippe M. Duquette Richard Marcotte Lam Leduy Sayeh Davoudi Nathalie Lamarche-Vane Angelo Iulianella Alain Nepveu 《The Journal of biological chemistry》2015,290(37):22520-22531
CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair. 相似文献
3.
Netanel Tzarum Nadav Komornik Dorin Ben Chetrit David Engelberg Oded Livnah 《The Journal of biological chemistry》2013,288(27):19537-19547
Signaling processes are primarily promoted by molecular recognition and corresponding protein-protein interactions. One of the key eukaryotic signaling pathways is the MAP kinase cascade involved in vital cellular processes such as cell proliferation, differentiation, apoptosis, and stress response. The principle recognition site of MAP kinases, the common docking (CD) region, forms selective interactions with substrates, upstream activators, and phosphatases. A second docking site, defined as the DEF site interaction pocket (DEF pocket), is formed subsequent to ERK2 and p38α activation. Both crystal structures of p38α in its dually phosphorylated form and of intrinsically active mutants showed the DEF pocket, giving motivation for studying its role in substrate activation and selectivity. Mutating selected DEF pocket residues significantly decreased the phosphorylation levels of three p38α substrates (ATFII, Elk-1, and MBP) with no apparent effect on the phosphorylation of MK2 kinase. Conversely, mutating the CD region gave the opposite effect, suggesting p38α substrates can be classified into DEF-dependent and DEF-independent substrates. In addition, mutating DEF pocket residues decreased the autophosphorylation capability of intrinsically active p38α mutants, suggesting DEF-mediated trans-autophosphorylation in p38α. These results could contribute to understanding substrate selectivity of p38α and serve as a platform for designing p38α-selective DEF site blockers, which partially inhibit p38α binding DEF-dependent substrates, whereas maintaining its other functions intact. In this context, preliminary results using synthetic peptides reveal significant inhibition of substrate phosphorylation by activated p38α. 相似文献
4.
Joseph M. Dahl Hongyun Wang José M. Lázaro Margarita Salas Kate R. Lieberman 《The Journal of biological chemistry》2014,289(10):6350-6361
The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn2+ rather than Mg2+. The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture. 相似文献
5.
Timofeyeva NA Koval VV Ishchenko AA Saparbaev MK Fedorova OS 《Biochemistry. Biokhimii?a》2011,76(2):273-281
Human major apurinic/apyrimidinic endonuclease (APE1) is a multifunctional enzyme that plays a central role in DNA repair
through the base excision repair (BER) pathway. Besides BER, APE1 is involved in an alternative nucleotide incision repair
(NIR) pathway that bypasses glycosylases. We have analyzed the conformational dynamics and the kinetic mechanism of APE1 action
in the NIR pathway. For this purpose we recorded changes in the intensity of fluorescence of 2-aminopurine located in two
different positions in a substrate containing dihydrouridine (DHU) during the interaction of the substrate with the enzyme.
The enzyme was found to change its conformation within the complex with substrate and also within the complex with the reaction
product, and the release of the enzyme from the complex with the product seemed to be the limiting stage of the enzymatic
process. The rate constants of the catalytic cleavage of DHU-containing substrates by APE1 were comparable with the appropriate
rate constants for substrates containing apurinic/apyrimidinic site or tetrahydrofuran residue, which suggests that NIR is
a biologically important process. 相似文献
6.
Specificity determinants in phosphoinositide dephosphorylation: crystal structure of an archetypal inositol polyphosphate 5-phosphatase 总被引:9,自引:0,他引:9
Inositol polyphosphate 5-phosphatases are central to intracellular processes ranging from membrane trafficking to Ca(2+) signaling, and defects in this activity result in the human disease Lowe syndrome. The 1.8 resolution structure of the inositol polyphosphate 5-phosphatase domain of SPsynaptojanin bound to Ca(2+) and inositol (1,4)-bisphosphate reveals a fold and an active site His and Asp pair resembling those of several Mg(2+)-dependent nucleases. Additional loops mediate specific inositol polyphosphate contacts. The 4-phosphate of inositol (1,4)-bisphosphate is misoriented by 4.6 compared to the reactive geometry observed in the apurinic/apyrimidinic endonuclease 1, explaining the dephosphorylation site selectivity of the 5-phosphatases. Based on the structure, a series of mutants are described that exhibit altered substrate specificity providing general determinants for substrate recognition. 相似文献
7.
Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase 总被引:1,自引:0,他引:1
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Kuznetsov NA Koval VV Zharkov DO Nevinsky GA Douglas KT Fedorova OS 《Nucleic acids research》2005,33(12):3919-3931
Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG. 相似文献
8.
Takayoshi Tagami Keitaro Yamashita Masayuki Okuyama Haruhide Mori Min Yao Atsuo Kimura 《The Journal of biological chemistry》2013,288(26):19296-19303
Sugar beet α-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher kcat/Km values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific α-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 Å resolution. The active site pocket of SBG is formed by a (β/α)8 barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (β/α)8 barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis. 相似文献
9.
A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches. 相似文献
10.
Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme in the base excision repair (BER) and nucleotide incision repair (NIR) pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt) protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5'-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (F, tetrahydrofuran) containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU) containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg(2+) concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP) site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage of DHU- and AP-substrates. 相似文献
11.
Kristina Lakomek Achim Dickmanns Lars Schomacher Ralf Ficner 《Journal of molecular biology》2010,399(4):604-617
The reliable repair of pre-mutagenic U/G mismatches that originated from hydrolytic cytosine deamination is crucial for the maintenance of the correct genomic information. In most organisms, any uracil base in DNA is attacked by uracil DNA glycosylases (UDGs), but at least in Methanothermobacter thermautotrophicus ΔH, an alternative strategy has evolved. The exonuclease III homologue Mth212 from the thermophilic archaeon M. thermautotrophicus ΔH exhibits a DNA uridine endonuclease activity in addition to the apyrimidinic/apurinic site endonuclease and 3′ → 5′exonuclease functions. Mth212 alone compensates for the lack of a UDG in a single-step reaction thus substituting the two-step pathway that requires the consecutive action of UDG and apyrimidinic/apurinic site endonuclease.In order to gain deeper insight into the structural basis required for the specific uridine recognition by Mth212, we have characterized the enzyme by means of X-ray crystallography. Structures of Mth212 wild-type or mutant proteins either alone or in complex with DNA substrates and products have been determined to a resolution of up to 1.2 Å, suggesting key residues for the uridine endonuclease activity. The insertion of the side chain of Arg209 into the DNA helical base stack resembles interactions observed in human UDG and seems to be crucial for the uridine recognition. In addition, Ser171, Asn153, and Lys125 in the substrate binding pocket appear to have important functions in the discrimination of aberrant uridine against naturally occurring thymidine and cytosine residues in double-stranded DNA. 相似文献
12.
《DNA Repair》2014
Mammalian AP endonuclease 1 is a pivotal enzyme of the base excision repair pathway acting on apurinic/apyrimidinic sites. Previous structural and biochemical studies showed that the conserved Asn-212 residue is important for the enzymatic activity of APE1. Here, we report a comprehensive pre-steady-state kinetic analysis of two APE1 mutants, each containing amino acid substitutions at position 212, to ascertain the role of Asn-212 in individual steps of the APE1 catalytic mechanism. We applied the stopped-flow technique for detection of conformational transitions in the mutant proteins and DNA substrates during the catalytic cycle, using fluorophores that are sensitive to the micro-environment. Our data indicate that Asn-212 substitution by Asp reduces the rate of the incision step by ∼550-fold, while Ala substitution results in ∼70,000-fold decrease. Analysis of the binding steps revealed that both mutants continued to rapidly and efficiently bind to abasic DNA containing the natural AP site or its tetrahydrofuran analogue (F). Moreover, transient kinetic analysis showed that N212A APE1 possessed a higher binding rate and a higher affinity for specific substrates compared to N212D APE1. Molecular dynamics (MD) simulation revealed a significant dislocation of the key catalytic residues of both mutant proteins relative to wild-type APE1. The analysis of the model structure of N212D APE1 provides evidence for alternate hydrogen bonding between Asn-212 and Asp-210 residues, whereas N212A possesses an extended active site pocket due to Asn removal. Taken together, these biochemical and MD simulation results indicate that Asn-212 is essential for abasic DNA incision, but is not crucial for effective recognition/binding. 相似文献
13.
Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism.
The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication.
Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding
to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with
MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative
damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of
the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies
have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of strated the substrate
recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base
flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the
repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis,
an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch
BER (2–6-nucleotide patch size) pathways need AP endonuclease to generate a 3′ hydroxyl group but require different sets of
enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER.
It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions.
The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis. 相似文献
14.
Up-regulation of base excision repair activity for 8-hydroxy-2'-deoxyguanosine in the mouse brain after forebrain ischemia-reperfusion 总被引:5,自引:0,他引:5
The repair enzyme 8-oxoguanine glycosylase/ apyrimidinic/apurinic lyase (OGG) removes 8-hydroxy-2'deoxyguanosine (oh8dG) in human cells. Our goal was to examine oh8dG-removing activity in the cell nuclei of male C57BL/6 mouse brains treated with either forebrain ischemia-reperfusion (FblR) or sham operations. We found that the OGG activity in nuclear extracts, under the condition in which other nucleases did not destroy the oligodeoxynucleotide duplex, excised oh8dG with the greatest efficiency on the oligodeoxynucleotide duplex containing oh8dG/dC and with less efficiency on the heteroduplex containing oh8dG/dT, oh8dG/dG, or oh8dG/dA. This specificity was the same as for the recombinant type 1 OGG (OGG1) of humans. We observed that the OGG1 peptide and its activity in the mouse brain were significantly increased after 90 min of ischemia and 20-30 min of reperfusion. The increase in the protein level and in the activity of brain OGG1 correlated positively with the elevation of FblR-induced DNA lesions in an indicator gene (the c-fos gene) of the brain. The data suggest a possibility that the OGG1 protein may excise oh8dG in the mouse brain and that the activity of OGG1 may have a functional role in reducing oxidative gene damage in the brain after FblR. 相似文献
15.
Vlatko Stojanoski Dar-Chone Chow Liya Hu Banumathi Sankaran Hiram F. Gilbert B. V. Venkataram Prasad Timothy Palzkill 《The Journal of biological chemistry》2015,290(16):10382-10394
β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism. 相似文献
16.
Dolores Córdoba‐Cañero Teresa Roldán‐Arjona Rafael R. Ariza 《The Plant journal : for cell and molecular biology》2014,79(5):824-834
Oxidation of guanine in DNA generates 7,8‐dihydro‐8‐oxoguanine (8‐oxoG), an ubiquitous lesion with mutagenic properties. 8‐oxoG is primarily removed by DNA glycosylases distributed in two families, typified by bacterial Fpg proteins and eukaryotic Ogg1 proteins. Interestingly, plants possess both Fpg and Ogg1 homologs but their relative contributions to 8‐oxoG repair remain uncertain. In this work we used Arabidopsis cell‐free extracts to monitor 8‐oxoG repair in wild‐type and mutant plants. We found that both FPG and OGG1 catalyze excision of 8‐oxoG in Arabidopsis cell extracts by a DNA glycosylase/lyase mechanism, and generate repair intermediates with blocked 3′‐termini. An increase in oxidative damage is detected in both nuclear and mitochondrial DNA from double fpg ogg1 mutants, but not in single mutants, which suggests that a single deficiency in one of these DNA glycosylases may be compensated by the other. We also found that the DNA 3′‐phosphatase ZDP (zinc finger DNA 3′‐phosphoesterase) and the AP(apurinic/apyirmidinic) endonuclease ARP(apurinic endonuclease redox protein) are required in the 8‐oxoG repair pathway to process the 3′‐blocking ends generated by FPG and OGG1. Furthermore, deficiencies in ZDP and/or ARP decrease germination ability after seed deteriorating conditions. Altogether, our results suggest that Arabidopsis cells use both FPG and OGG1 to repair 8‐oxoG in a pathway that requires ZDP and ARP in downstream steps. 相似文献
17.
Asparagine 212 is essential for abasic site recognition by the human DNA repair endonuclease HAP1. 总被引:2,自引:0,他引:2
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HAP1 is a divalent cation-dependent endonuclease from human cells with specificity for apurinic/apyrimidinic (AP) sites in DNA. Extraction of the essential metal ion from purified HAP1 stabilized its binding to an oligonucleotide containing a single AP site, permitting AP site binding studies to be undertaken using gel retardation assays. Binding of HAP1 to such an oligonucleotide was dependent upon the presence of an AP site. Previous structural and modelling studies have suggested a role for Asn212 (Asn153 in exonuclease III, the bacterial homologue of HAP1) in substrate recognition. Substitution of alanine for Asn212 abolished the AP endonuclease activity of purified recombinant HAP1 protein. More conservative substitutions of aspartate or glutamine for Asn212 still led to a reduction in specific activity of at least 300-fold. Moreover, none of the three Asn212 substitution mutants of HAP1 possessed detectable AP site binding activity in vitro. This study indicates that chelation of the active site metal ion in HAP1 stabilizes the complex of the protein with AP sites and identifies an active site asparagine residue as an important component of AP site recognition by the HAP1 protein. 相似文献
18.
Kuznetsov NA Koval VV Nevinsky GA Douglas KT Zharkov DO Fedorova OS 《The Journal of biological chemistry》2007,282(2):1029-1038
7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower beta-elimination reaction at the 3'-side of the resulting abasic site. Many features of OGG1 mechanism, including its low beta-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of beta-elimination rather than pre-excision dynamics of DNA and the enzyme. 相似文献
19.
ES Vik I Alseth M Forsbring IH Helle I Morland L Luna M Bjørås B Dalhus 《DNA Repair》2012,11(9):766-773
Base excision repair of oxidized DNA in human cells is initiated by several DNA glycosylases with overlapping substrate specificity. The human endonuclease VIII homologue NEIL1 removes a broad spectrum of oxidized pyrimidine and purine lesions. In this study of NEIL1 we have identified several key residues, located in three loops lining the DNA binding cavity, important for lesion recognition and DNA glycosylase/AP lyase activity for oxidized bases in double-stranded and single-stranded DNA. Single-turnover kinetics of NEIL1 revealed that removal of 5-hydroxycytosine (5-OHC) and 5-hydroxyuracil (5-OHU) is ~25 and ~10-fold faster in duplex DNA compared to single-stranded DNA, respectively, and also faster than removal of dihydrothymine (DHT) and dihydrouracil (DHU), both in double-stranded and single-stranded DNA. NEIL1 excised 8-oxoguanine (8-oxoG) only from double-stranded DNA and analysis of site-specific mutants revealed that Met81, Arg119 and Phe120 are essential for removal of 8-oxoG. Further, several arginine and histidine residues located in the loop connecting the two β-strands forming the zincless finger motif and projecting into the DNA major groove, were shown to be imperative for lesion processing for both single- and double-stranded substrates. Trapping experiments of active site mutants revealed that the N-terminal Pro2 and Lys54 can alternate to form a Schiff-base complex between the protein and DNA. Hence, both Pro2 and Lys54 are involved in the AP lyase activity. While wildtype NEIL1 activity almost exclusively generated a δ-elimination product when processing single-stranded substrates, substitution of Lys54 changed this in favor of a β-elimination product. These results suggest that Pro2 and Lys54 are both essential for the concerted action of the β,δ-elimination in NEIL1. 相似文献
20.
Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair 总被引:17,自引:10,他引:7
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8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG·C-containing duplex oligo with a very low apparent kcat of 0.1 min–1 at 37°C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis–Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased ~5-fold and Michaelis–Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP·C pair (Kd ~ 2.8 nM) was substantially higher than for its substrate 8-oxoG·C pair (Kd ~ 23.4 nM) and the affinity for its final β-elimination product was much lower (Kd ~ 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway. 相似文献