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The structure of cytoplasmic malate dehydrogenase has been partially refined by crystallographic least squares methods. Using x-ray phases based on the refined coordinates, analysis of the resultant electron density maps has led to a new model of cytoplasmic malate dehydrogenase and a tentative "x-ray sequence." The two crystallographically independent subunits comprising the dimeric enzyme are nearly identical in structure and are related to each other by roughly 2-fold rotational symmetry. The best fit of the molecular structure of cytoplasmic malate dehydrogenase to that of lactate dehydrogenase has been obtained by least squares methods. The active sites of these two enzymes contain similarly oriented His-Asp pairs linked by a hydrogen bond which may function as a proton relay system during catalysis. This pair could also provide an explanation for the relatively stronger binding by cytoplasmic malate dehydrogenase and lactate dehydrogenase of NADH versus NAD. Similar His-Asp pairs have been observed in the serine proteases, thermolysin, and phospholipase A2, and the His-Asp pair may play a similar functional role in all of these enzymes. 相似文献
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Wilson DM 《Journal of molecular biology》2003,330(5):1027-1037
Ape1 is the major human abasic endonuclease, initiating repair of this common DNA lesion by incising the phosphodiester backbone 5' to the damage site. This enzyme also functions in specific contexts to excise 3'-blocking termini, e.g. phosphate and phosphoglycolate residues, from DNA. Recently, the comparatively "minor" 3' to 5' exonuclease activity of Ape1 was found to contribute to the excision of certain 3'-mismatched nucleotides. In this study, I characterize more thoroughly the 3'-nuclease properties of Ape1 and define the effects of specific DNA determinants on this function. Data within shows that Ape1 is a non- or poorly processive exonuclease, which degrades one nucleotide gap, 3'-recessed, and nicked DNAs, but exhibits no detectable activity on blunt end or single-stranded DNA. A 5'-phosphate, compared to a 5'-hydroxyl group, reduced Ape1 degradation activity roughly tenfold, suggesting that the biological impact of certain DNA single strand breaks may be influenced by the terminal chemistry. In the context of a base excision repair-like DNA intermediate, a 5'-abasic residue exerted an about tenfold attenuation on the 3' to 5' exonuclease efficiency of Ape1. A 3'-phosphate group had little impact on Ape1 exonuclease activity, and oligonucleotides harboring these blocking termini were activated by Ape1 for DNA polymerase beta extension. Ape1 was also found to remove 3'-tyrosyl residues from 3'-recessed and nicked DNAs, suggesting a potential role in processing covalent topoisomerase I-DNA intermediates formed during chromosome relaxation. While exhibiting preferential excision of thymine in a T:G mismatch context, Ape1 was unable to degrade a triple 3'-thymine mispair. However, Ape1 was able to excise double nucleotide mispairs, apparently through a novel 3'-flap-type endonuclease activity, again activating these substrates for polymerase beta extension. 相似文献
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Apurinic/apyrimidinic (AP) endonuclease Ape1 is a key enzyme in the mammalian base excision repair pathway that corrects AP sites in the genome. Ape1 cleaves the phosphodiester bond immediately 5' to AP sites through a hydrolytic reaction involving a divalent metal co-factor. Here, site-directed mutagenesis, chemical footprinting techniques, and molecular dynamics simulations were employed to gain insights into how Ape1 interacts with its metal cation and AP DNA. It was found that Ape1 binds predominantly to the minor groove of AP DNA, and that residues R156 and Y128 contribute to protein-DNA complex stability. Furthermore, the Ape1-AP DNA footprint does not change along its reaction pathway upon active-site coordination of Mg(2+) or in the presence of DNA polymerase beta (polbeta), an interactive protein partner in AP site repair. The DNA region immediately 5' to the abasic residue was determined to be in close proximity to the Ape1 metal-binding site. Experimental evidence is provided that amino acid residues E96, D70, and D308 of Ape1 are involved in metal coordination. Molecular dynamics simulations, starting from the active site of the Ape1 crystal structure, suggest that D70 and E96 bind directly to the metal, while D308 coordinates the cation through the first hydration shell. These studies define the Ape1-AP DNA interface, determine the effect of polbeta on the Ape1-DNA interaction, and reveal new insights into the Ape1 active site and overall protein dynamics. 相似文献
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1H NMR investigation of the conformational states of DNA in nucleosome core particles. 总被引:3,自引:3,他引:0
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In this study 1H NMR has been used to investigate the conformational state of DNA in nucleosome core particles. The nucleosome core particles exhibit partially resolved low field (10-15 ppm) spectra due to imino protons in Watson-Crick base pairs (one resonance per GC or AT base pair). To a first approximation, the spectrum is virtually identical with that of protein-free 140 base pair DNA, and from this observation we draw two important conclusions: (i) Since the low field spectra of DNA are known to be sensitive to conformation, the conformation of DNA in the core particles is essentially the same as that of free DNA (presumably B-form), (ii) since kinks occurring at a frequency at 1 in 10 or 1 in 20 base pairs would result in a core particle spectrum different from that of free DNA we find no NMR evidence supporting either the Crick-Klug or the Sobell models for kinking DNA around the core histones. Linewidth considerations indicate that the rotational correlation time for the core particles is approximately 1.5 X 10(-7) sec, whereas the end-over-end tumbling time of the free 140 base pair DNA is 3 X 10(-7) sec. 相似文献
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Ionizing radiation (IR) and bleomycin (BLM) are used to treat various types of cancers. Both agents generate cytotoxic double strand breaks (DSB) and abasic (apurinic/apyrimidinic (AP)) sites in DNA. The human AP endonuclease Ape1 acts on abasic or 3'-blocking DNA lesions such as those generated by IR or BLM. We examined the effect of siRNA-mediated Ape1 suppression on DNA repair and cellular resistance to IR or BLM in human B-lymphoblastoid TK6 cells and HCT116 colon tumor cells. Partial Ape1 deficiency (~30% of normal levels) sensitized cells more dramatically to BLM than to IR cytotoxicity. In both cases, expression of the unrelated yeast AP endonuclease, Apn1, largely restored resistance. Ape1 deficiency increased DNA AP site accumulation due to IR treatment but reduced the number of DSB. In contrast, for BLM, there were more DSB under Ape1 deficiency, with little change in the accumulation of AP sites. Although the role of Ape1 in generating DSB was greater for IR, the enzyme facilitated removal of AP sites, which may mitigate the cytotoxic effects of IR. In contrast, BLM generates scattered AP sites, and the DSB have 3'-phosphoglycolate termini that require Ape1 processing. These DSB persist under Ape1 deficiency. Apoptosis induced by BLM (but not by IR) under Ape1 deficiency was partially p53-dependent, more dramatically in TK6 than HCT116 cells. Thus, Ape1 suppression or inhibition may be a more efficacious adjuvant for BLM than for IR cancer therapy, particularly for tumors with a functional p53 pathway. 相似文献
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Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair. 相似文献
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Daviet S Couvé-Privat S Gros L Shinozuka K Ide H Saparbaev M Ishchenko AA 《DNA Repair》2007,6(1):8-18
Most common point mutations occurring spontaneously or induced by ionizing radiation are C-->T transitions implicating cytosine as the target. Oxidative cytosine derivatives are the most abundant and mutagenic DNA damage induced by oxidative stress. Base excision repair (BER) pathway initiated by DNA glycosylases is thought to be the major pathway for the removal of these lesions. However, in alternative nucleotide incision repair (NIR) pathway the apurinic/apyrimidinic (AP) endonucleases incise DNA duplex 5' to an oxidatively damaged base in a DNA glycosylase-independent manner. Here, we characterized the substrate specificity of human major AP endonuclease, Ape1, towards 5-hydroxy-2'-deoxycytidine (5ohC) and alpha-anomeric 2'-deoxycytidine (alphadC) residues. The apparent kinetic parameters of the reactions suggest that Ape1 and the DNA glycosylases/AP lyases, hNth1 and hNeil1 repair 5ohC with a low efficiency. Nevertheless, due to the extremely high cellular concentration of Ape1, NIR was the major activity towards 5ohC in cell-free extracts. To address the physiological role of NIR function, we have characterized naturally occurring Ape1 variants including amino acids substitutions (E126A, E126D and D148E) and N-terminal truncated forms (NDelta31, NDelta35 and NDelta61). As expected, all Ape1 mutants had proficient AP endonuclease activity, however, truncated forms showed reduced NIR and 3'-->5' exonuclease activities indicating that these two functions are genetically linked and governed by the same amino acid residues. Furthermore, both Ape1-catalyzed NIR and 3'-->5' exonuclease activities generate a single-strand gap at the 5' side of a damaged base but not at an AP site in duplex DNA. We hypothesized that biochemical coupling of the nucleotide incision and exonuclease degradation may serve to remove clustered DNA damage. Our data suggest that NIR is a backup system for the BER pathway to remove oxidative damage to cytosines in vivo. 相似文献
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The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta. To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta. However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta. 相似文献
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Aishima J Gitti RK Noah JE Gan HH Schlick T Wolberger C 《Nucleic acids research》2002,30(23):5244-5252
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The Ape1 protein initiates the repair of apurinic/apyrimidinic sites during mammalian base excision repair (BER) of DNA. Ape1 catalyzes hydrolysis of the 5'-phosphodiester bond of abasic DNA to create nicks flanked by 3'-hydroxyl and 5'-deoxyribose 5-phosphate (dRP) termini. DNA polymerase (pol) beta catalyzes both DNA synthesis at the 3'-hydroxyl terminus and excision of the 5'-dRP moiety prior to completion of BER by DNA ligase. During BER, Ape1 recruits pol beta to the incised apurinic/apyrimidinic site and stimulates 5'-dRP excision by pol beta. The activities of these two enzymes are thus coordinated during BER. To examine further the coordination of BER, we investigated the ability of Ape1 to modulate the deoxynucleotidyltransferase and 5'-dRP lyase activities of pol beta. We report here that Ape1 stimulates 5'-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. We also demonstrate a second mechanism, independent of Ape1, in which conditions that support DNA synthesis by pol beta also enhance 5'-dRP excision. Ape1 modulates the gap-filling activity of pol beta by specifically inhibiting synthesis on an incised abasic substrate but not on single-nucleotide gapped DNA. In contrast to the wild-type Ape1 protein, a catalytically impaired mutant form of Ape1 did not affect DNA synthesis by pol beta. However, this mutant protein retained the ability to stimulate 5'-dRP excision by pol beta. Simultaneous monitoring of 5'-dRP excision and DNA synthesis by pol beta demonstrated that the 5'-dRP lyase activity lags behind the polymerase activity despite the coordination of these two steps by Ape1 during BER. 相似文献
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Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs
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DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease. 相似文献
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We have previously shown that a short 16 base pair DNA oligomer can accommodate a B-Z conformational junction [Sheardy, R. D., & Winkle, S. A. (1989) Biochemistry 28, 720-725]. Results from 1H NMR studies indicated that only three base pairs were involved in the junction and that one of these base pairs was highly distorted. Being interested in the nature of this distortion, we constructed DNA oligomers which have the potential to contain multiple B-Z junctions for polyacrylamide electrophoretic studies. We report that the mobilities displayed by these molecules through acrylamide gels in the absence and presence of cobalt suggest that these molecules run shorter than they actually are. This anomalous migration may be due to structural/dynamic properties of the DNA helix manifested by the periodic distortions of the potential B-Z junctions. 相似文献