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

---

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

The DNA repair enzyme uracil DNA glycosylase (UDG) hydrolyzes the glycosidic bond of deoxyuridine in DNA by a remarkable mechanism involving formation of a positively charged oxacarbenium ion-uracil anion intermediate. We have proposed that the positively charged intermediate is stabilized by being sandwiched between the combined negative charges of the anionic uracil leaving group and a conserved aspartate residue that are located on opposite faces of the sugar ring. Here we establish that a duplex DNA oligonucleotide containing a cationic 1-aza-deoxyribose (I) oxacarbenium ion mimic is a potent inhibitor of UDG that binds tightly to the enzyme-uracil anion (EU(-)) product complex (K(D) of EU(-) = 110 pm). The tight binding of I to the EU(-) complex results from its extremely slow off rate (k(off) = 0.0008 s(-1)), which is 25,000-fold slower than substrate analogue DNA. Removal of Asp(64) and His(187), which are involved in stabilization of the cationic sugar and the anionic uracil leaving group, respectively, specifically weakens binding of I to the UDG-uracil complex by 154,000-fold, without significantly affecting substrate or product binding. These results suggest that electrostatic effects can effectively stabilize such an intermediate by at least -7 kcal/mol, without leading to anticatalytic stabilization of the substrate and products.
...
PMID:Probing the limits of electrostatic catalysis by uracil DNA glycosylase using transition state mimicry and mutagenesis. 1185 82

We have investigated the inhibition of the DNA repair enzyme uracil DNA glycosylase (UDG) by an 11-mer oligonucleotide (AIA) containing a cationic 1-aza-deoxyribose (I) residue designed to be a stable mimic of the high-energy oxacarbenium ion reaction intermediate [Werner, R. M., and Stivers, J. T. (2000) Biochemistry 39, 14054-14064]. Inhibition kinetics and direct binding studies indicate that AIA binds weakly to the free enzyme (K(D) = 2 microM) but binds 4000-fold more tightly to the enzyme-uracil anion (EU) product complex (K(D) = 500 pM). The importance of the positive charge on the 1-nitrogen in binding is established by the observation that AIA binds >30 000-fold more tightly to the EU complex than the corresponding neutral tetrahydrofuran (F) abasic site product analogue (AFA). The unusual inhibition mechanism for AIA results in a time dependence that resembles slow-onset inhibition even though the apparent on-rate of the inhibitor for the EU(-) binary product complex is moderate (1 microM(-1) x s(-1)). Accordingly, the low K(D) of AIA for the EU complex is largely due its very slow off-rate (5 x 10(-4) x s(-1)). These results support previous kinetic isotope effect measurements that indicate UDG stabilizes a discrete oxacarbenium ion-uracil anion intermediate. This oxacarbenium ion mimic represents the tightest binding inhibitor of UDG yet identified.
...
PMID:Inhibition of uracil DNA glycosylase by an oxacarbenium ion mimic. 1203 46

The single-stranded DNA-binding proteins (SSBs) are vital to virtually all DNA functions. Here, we report on the biochemical properties of SSB from a fast-growing mycobacteria, Mycobacterium smegmatis, and the interaction of the homotetrameric SSBs with uracil DNA glycosylases (UDGs) from M. smegmatis (Msm), Mycobacterium tuberculosis (Mtu) and Escherichia coli (Eco). UDG is a crucial DNA repair enzyme, which removes the promutagenic uracil residues. MsmSSB stimulates activity of the homologous Msm UDG and of the heterologous Mtu-, and Eco-UDGs. On the contrary, while the MtuSSB stimulates the Mtu UDG, it inhibits the other two UDGs. Although the MsmSSB shares 84% identity with MtuSSB, the two are strikingly different, in that MsmSSB contains a glycine-rich segment (11 out of 13 residues) in the spacer connecting the N-terminal DNA-binding domain with the C-terminal acidic tail. While the DNA-binding properties of MsmSSB, such as its affinity to oligomeric DNA, requirement of minimum size DNA and the modes of interaction are indistinguishable from those of Eco-, and Mtu-SSBs, it is unclear if the glycine-rich segment confers structural advantage to MsmSSB, responsible for its stimulatory effect on all UDGs tested. More importantly, by using a small polypeptide inhibitor of UDGs, and the deletion mutants of SSBs, we suggest that the C-terminal acidic tail of the SSBs interacts within the DNA-binding groove of the UDGs, and propose a role for SSBs in the recruitment of UDGs to the damaged DNA.
...
PMID:Biochemical properties of single-stranded DNA-binding protein from Mycobacterium smegmatis, a fast-growing mycobacterium and its physical and functional interaction with uracil DNA glycosylases. 1208 15

The DNA repair enzyme uracil DNA glycosylase (UDG) locates unwanted uracil bases in genomic DNA using a remarkable base-flipping mechanism in which the entire deoxyuridine nucleotide is rotated from the DNA base stack into the enzyme active site. Enzymatic base flipping has been described as a three-step process involving phosphodiester backbone pinching, base extrusion through active pushing and plugging by a leucine side chain that inserts in the DNA minor groove, and, finally, pulling by hydrogen-bonding groups that interact with the extrahelical base. Here we employ mutagenesis in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the "pinch, push, plug, and pull" base-flipping mechanism. Our results show that these mutant enzymes are capable of flipping the uracil base from the duplex, but that many of these mutations prevent a subsequent induced fit conformational step in which catalytic groups of UDG dock with the flipped-out base. These studies support our previous model for base flipping in which a conformational gating step closely follows base extrusion from the DNA duplex [Stivers, J. T., et al. (1999) Biochemistry 38, 952-963]. A model that accounts for the temporal and functional roles of these side chain interactions along the reaction pathway for base flipping is presented.
...
PMID:Mutational analysis of the base-flipping mechanism of uracil DNA glycosylase. 1222 Jan 89

The mechanism by which brief episodes of cerebral ischemia confer protection (tolerance) against subsequent prolonged ischemic challenges remains unclear, but may involve upregulation of cell injury repair capability. The mitochondrion is a key site for the regulation of cell death pathways, and damage to mitochondrial genes has been linked to a number of neurologic diseases and aging. Therefore, the authors examined the response of the DNA base excision repair (BER) pathway in rat brain mitochondria after either brief (tolerance-inducing) or prolonged (injury-producing) focal cerebral ischemia. Brief (30-minute) middle cerebral artery occlusion (MCAO) induced mild oxidative mitochondrial DNA damage and initiated a prolonged (up to 72-hour) activation above control levels of the principal enzymes of the mitochondrial BER pathway, including uracil DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease, DNA polymerase-gamma, and DNA ligase. In contrast, prolonged (100-minute MCAO) ischemia induced more substantial mitochondrial oxidative DNA damage whereas elevation of BER activity was transient (approximately 1 hour), declining to less than control levels over the course of 4 to 72 hours. These data reveal the differences in BER capacity after brief or prolonged ischemia, which may contribute to the neuron's ability to resist subsequent ischemic insults.
...
PMID:Upregulation of mitochondrial base-excision repair capability within rat brain after brief ischemia. 1250 94

The reaction catalyzed by the DNA repair enzyme uracil DNA glycosylase (UDG) proceeds through an unprecedented stepwise mechanism involving a positively charged oxacarbenium ion sugar and uracil anion leaving group. Here we use a novel approach to evaluate the catalytic contribution of electrostatic interactions between four essential phosphodiester groups of the DNA substrate and the cationic transition state. Our strategy was to substitute each of these phosphate groups with an uncharged (R)- or (S)-methylphosphonate linkage (MeP). We then compared the damaging effects of these methylphosphonate substitutions on catalysis with their damaging effects on binding of a cationic 1-azadeoxyribose (1-aza-dR(+)) oxacarbenium ion analogue to the UDG-uracil anion binary complex. A plot of log k(cat)/K(m) for the series of MeP-substituted substrates against log K(D) for binding of the 1-aza-dR(+) inhibitors gives a linear correlation of unit slope, confirming that the electronic features of the transition state resemble that of the 1-aza-dR(+), and that the anionic backbone of DNA is used in transition state stabilization. We estimate that all of the combined phosphodiester interactions with the substrate contribute 6-8 kcal/mol toward lowering the activation barrier, a stabilization that is significant compared to the 16 kcal/mol catalytic power of UDG. However, unlike groups of the enzyme that selectively stabilize the charged transition state by an estimated 7 kcal/mol, these phosphodiester groups also interact strongly in the ground state. To our knowledge, these results provide the first experimental evidence for electrostatic stabilization of a charged enzymatic transition state and intermediate using the anionic backbone of DNA.
...
PMID:Powering DNA repair through substrate electrostatic interactions. 1259 May 78

A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a "preflipped" cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.
...
PMID:Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair. 1272 63

The DNA repair enzyme uracil DNA glycosylase has been crystallized with a cationic 1-aza-2'-deoxyribose-containing DNA that mimics the ultimate transition state of the reaction in which the water nucleophile attacks the anomeric center of the oxacarbenium ion-uracil anion reaction intermediate. Comparison with substrate and product structures, and the previous structure of the intermediate determined by kinetic isotope effects, reveals an exquisite example of geometric strain, least atomic motion, and electrophile migration in biological catalysis. This structure provides a rare opportunity to reconstruct the detailed structural transformations that occur along an enzymatic reaction coordinate.
...
PMID:Electrostatic guidance of glycosyl cation migration along the reaction coordinate of uracil DNA glycosylase. 1458 Jan 90

Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.
...
PMID:Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the virus mutation rate and for replication in macrophages. 1509 17

The DNA repair enzyme uracil DNA glycosylase (UDG) utilizes base flipping to recognize and remove unwanted uracil bases from the genome but does not react with its structural congener, thymine, which differs by a single methyl group. Two factors that determine whether an enzyme flips a base from the duplex are its shape and hydrogen bonding properties. To probe the role of these factors in uracil recognition by UDG, we have synthesized a DNA duplex that contains a single difluorophenyl (F) nucleotide analogue that is an excellent isostere of uracil but possesses no hydrogen bond donor or acceptor groups. By using binding affinity measurements, solution (19)F NMR, and solid state (31)P[(19)F] rotational-echo double-resonance (REDOR) NMR measurements, we establish that UDG partially unstacks F from the duplex. However, due to the lack of hydrogen bonding groups that are required to support an open-to-closed conformational transition in UDG, F cannot stably dock in the UDG active site. We propose that F attains a metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathway and suggest structural models of the metastable state that are consistent with the REDOR NMR measurements.
...
PMID:Recognition of an unnatural difluorophenyl nucleotide by uracil DNA glycosylase. 1558 54


<< Previous 1 2 3 4 5 Next >>

---