Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

7-Deaza-2'-deoxyadenosine (1, c7Ad) and 3-deaza-2'-deoxyadenosine (2, c3Ad) have been incorporated into d(AAAAAA) tracts replacing dA at various positions within oligonucleotides. For this purpose suitably protected phosphonates have been prepared and oligonucleotides were synthesized on solid-phase. The oligomers were hybridized with their cognate strands. The duplexes were phosphorylated at OH-5' by polynucleotide kinase and self-ligated to multimers employing T4 DNA ligase. Oligomerized DNA-fragments were analyzed by polyacrylamide gel electrophoresis and the bending was determined from anomalies of electrophoretic mobility. Replacement of dA by c3Ad decreased the bending more than replacement by c7Ad. Reduction of bending was much stronger when the modified nucleosides replaced one or several dA residues at the 3'-site of an d(AAAAAA)-tract whereas replacement at the 5'-site showed no significant influence [1, 2].
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PMID:7-Deaza-2'-deoxyadenosine and 3-deaza-2'-deoxyadenosine replacing dA within d(A6)-tracts: differential bending at 3'- and 5'-junctions of d(A6).d(T6) and B-DNA. 163 Aug 98

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7, 8-dihydro-8-oxo-2'-deoxyguanosine: 2'-deoxyadenosine (OG:A) mismatches in DNA [Michaels et al. (1992) Proc. Natl. Acad. Sci. U.S. A. 89, 7022-7025]. MutY prevents mutations due to misincorporation of A opposite OG during DNA replication by removing the adenine base. This enzyme has significant sequence homology with the [4Fe-4S]2+ cluster-containing DNA repair enzyme, endonuclease III [Michaels et al. (1990) Nucleic AcidsRes. 18, 3841-3845]. In the present study, we have investigated the importance of cluster assembly in folding of MutY. MutY was denatured and then refolded in the presence or absence of ferrous and sulfide ions. Denatured MutY can refold in the presence of ferrous and sulfide ions to provide active enzyme. This suggests the cluster can self-assemble and that this process is facile in vitro. Interestingly, CD spectra and Tm measurements of MutY refolded with and without ferrous and sulfide ions are essentially identical, implying that assembly of the cluster is not required for MutY folding. Additionally, Tm measurements indicated that the [4Fe-4S]2+ cluster does not contribute significantly to the overall thermal stability of MutY. Refolded forms of MutY which lack the cluster are unable to perform the adenine glycosylase function and bind to DNA. However, these inactive folded forms regain activity by addition of ferrous and sulfide ions. This indicates that the Fe-S cluster may have a superficial location, allowing for its assembly after folding. More importantly, these results provide evidence that the presence of the [4Fe-4S]2+ cluster is critical for the specific recognition of substrate DNA necessary for the adenine glycosylase activity of MutY.
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PMID:A substrate recognition role for the [4Fe-4S]2+ cluster of the DNA repair glycosylase MutY. 957 64

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7, 8-dihydro-8-oxo-2'-deoxyguanosine:2'-deoxyadenosine (OG:A) mismatches in DNA [Michaels et al. (1992) Proc. Natl. Acad. Sci. U.S. A. 89, 7022-7025]. MutY prevents DNA mutations resulting from the misincorporation of A opposite OG by using N-glycosylase activity to remove the adenine base. An interesting feature of MutY is that it contains a [4Fe-4S]2+ cluster that has been shown to play an important role in substrate recognition [Porello, S. L., Cannon, M. J., David, S. S. (1998) Biochemistry 37, 6465-6475]. Herein, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, and alanine. The extent to which the various mutations reduce the levels of protein overexpression suggests that coordination of the [4Fe-4S]2+ cluster provides stability to MutY in vivo. The ability of the mutated enzymes to bind to a substrate analogue DNA duplex and their in vivo activity were evaluated. Remarkably, the effects are both substitution and position dependent. For example, replacement of cysteine 199 with histidine provides a mutated enzyme that is expressed at high levels and exhibits DNA binding and in vivo activity similar to the WT enzyme. These results suggest that histidine coordination to the iron-sulfur cluster may be accommodated at this position in MutY. In contrast, replacement of cysteine 192 with histidine results in less efficient DNA binding and in vivo activity compared to the WT enzyme without affecting levels of overexpression. The results from the site-directed mutagenesis suggest that the structural properties of the iron-sulfur cluster coordination domain are important for both substrate DNA recognition and the in vivo activity of MutY.
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PMID:Site-directed mutagenesis of the cysteine ligands to the [4Fe-4S] cluster of Escherichia coli MutY. 1035 11

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7, 8-dihydro-8-oxo-2'-deoxyguanosine:2'-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1-Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.
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PMID:Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain. 1113 26

The major DNA photoproduct of dormant, UV-irradiated Bacillus subtilis spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)]. During spore germination, SP is reversed to two intact thymines in situ by the DNA repair enzyme SP lyase, an S-adenosylmethionine (S-AdoMet)-dependent iron-sulfur ([Fe-S]) protein encoded by the splB gene. In the present work, cross-linking, SDS/PAGE, and size exclusion chromatography revealed that SplB protein dimerized when incubated with iron and sulfide under anaerobic reducing conditions. SplB isolated under aerobic conditions generated an EPR spectrum consistent with that of a partially degraded [3Fe-4S] center, and reduction of SplB with dithionite shifted the spectrum to that of a [4Fe-4S] center. Addition of S-AdoMet to SplB converted some of the [4Fe-4S] centers to an EPR-silent form consistent with electron donation to S-AdoMet. HPLC and electrospray ionization MS analyses showed that SP lyase cleaved S-AdoMet to generate 5'-deoxyadenosine. The results indicate that (i) SP lyase is a homodimer of SplB; (ii) dimer formation is coordinated by a [4Fe-4S] center; and (iii) the reduced [4Fe-4S] center is capable of donating electrons to S-AdoMet to generate a 5'-adenosyl radical that is then used for the in situ reversal of SP. Thus, SP lyase belongs to the "radical SAM" superfamily of enzymes that use [Fe-S] centers and S-AdoMet to generate adenosyl radicals to effect catalysis. SP lyase is unique in being the first and only DNA repair enzyme known to function via this novel enzymatic mechanism.
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PMID:The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase. 1147 Sep 12

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.
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PMID:Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster. 1190 May 36

MutY is an Escherichia coli DNA repair enzyme that binds to 8-oxo-G:A and G:A mismatches and catalyzes the deglycosylation of the mismatched 2'-deoxyadenosine. We have applied DNA-mediated charge transport to probe the interaction of MutY with its DNA substrate. Oligonucleotides synthesized with a tethered rhodium intercalator and guanine doublets placed before and after the MutY binding site are used to assay for base flipping activity by MutY. On the basis of this assay, we find no evidence that MutY uses progressive base flipping as a means to find its binding site; protein binding does not perturb long-range DNA charge transport. DNA-mediated charge transport can be utilized to promote protein-DNA cross-linking from a distance. Long-range oxidation of 8-oxo-G within the MutY binding site using tethered rhodium intercalators promoted cross-linking and yielded information on MutY side chains that interact with this base. On the basis of photooxidative cross-linking of the wild type but not K142A mutant, it is evident that, within the protein complex, lysine 142 makes important contacts with 8-oxo-G.
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PMID:DNA-mediated charge transport as a probe of MutY/DNA interaction. 1208 96

In vitro work in this laboratory has identified new DNA lesions resulting from further oxidation of a common biomarker of oxidative damage, 8-oxo-7,8-dihydroguanine (OG). The major product of oxidation of OG in a nucleoside, nucleotide, or single-stranded oligodeoxynucleotide using metal ions that act as one-electron oxidants is the new nucleoside derivative spiroiminodihydantoin (Sp). In duplex DNA an equilibrating mixture of two isomeric products, guanidinohydantoin (Gh) and iminoallantoin (Ia), is produced. These products are also formed by the overall four-electron oxidation of guanosine by photochemical processes involving O(2). DNA template strands containing either Sp or Gh/Ia generally acted as a block to DNA synthesis with the Klenow exo(-) fragment of pol I. However, when nucleotide insertion did occur opposite the lesions, only 2'-deoxyadenosine 5-triphosphate and 2'-deoxyguanine 5-triphosphate were used for primer extension. The Escherichia coli DNA repair enzyme Fpg was able to remove the Sp and Gh/Ia lesions from duplex DNA substrates, although the efficiency was depended on the base opposite the lesion.
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PMID:Structure and potential mutagenicity of new hydantoin products from guanosine and 8-oxo-7,8-dihydroguanine oxidation by transition metals. 1242 18

The DNA repair enzyme NEIL1 is a DNA glycosylase that is involved in the first step of base excision repair (BER) of oxidatively induced DNA damage. NEIL1 exhibits a strong preference for excision of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA with no specificity for 8-hydroxyguanine (8-OH-Gua). In this study, we report on the significant accumulation of (5'R)-8,5'-cyclo-2'-deoxyadenosine (R-cdA) and (5'S)-8,5'-cyclo-2'-deoxyadenosine (S-cdA) in liver DNA of neil1(-/-) mice that were not exposed to exogenous oxidative stress, while no accumulation of these lesions was observed in liver DNA from control or ogg1(-/-) mice. Significant accumulation of FapyGua was detected in liver DNA of both neil1(-/-) and ogg1(-/-) mice, while 8-OH-Gua accumulated in ogg1(-/-) only. Since R-cdA and S-cdA contain an 8,5'-covalent bond between the base and sugar moieties, they cannot be repaired by BER. There is evidence that these lesions are repaired by nucleotide excision repair (NER). Since the accumulation of R-cdA and S-cdA in neil1(-/-) mice strongly points to the failure of their repair, these data suggest that NEIL1 is involved in NER of R-cdA and S-cdA. Further studies aimed at elucidating the mechanism of action of NEIL1 in NER are warranted.
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PMID:Evidence for the involvement of DNA repair enzyme NEIL1 in nucleotide excision repair of (5'R)- and (5'S)-8,5'-cyclo-2'-deoxyadenosines. 2006 21

DNA lesions produced by aromatic isocyanates have an extra bulky group on the nucleotide bases, with the capability of forming stacking interaction within a DNA helix. In this work, we investigated the conformation of the 2'-deoxyadenosine and 2'-deoxycytidine derivatives tethering a phenyl or naphthyl group, introduced in a DNA duplex. The chemical modification experiments using KMnO4 and 1-cyclohexyl-3 -(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate have shown that the 2'-deoxycytidine lesions form the base pair with guanine while the 2'-deoxyadenosine lesions have less ability of forming the base pair with thymine in solution. Nevertheless, the kinetic analysis shows that these DNA lesions are compatible with DNA ligase and DNA polymerase reactions, as much as natural DNA bases. We suggest that the adduct lesions have a capability of adopting dual conformations, depending on the difference in their interaction energies between stacking of the attached aromatic group and base pairing through hydrogen bonds. It is also presented that the attached aromatic groups change their orientation by interacting with the minor groove binding netropsin, distamycin and synthetic polyamide. The nucleotide derivatives would be useful for enhancing the phenotypic diversity of DNA molecules and for exploring new non-natural nucleotides.
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PMID:Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules. 2387 56


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